WO2008025160A1 - Oxepane nucleosides and oligonucleotides, uses thereof and methods of making the same - Google Patents

Abstract

The present invention provides oxepane nucleosides, oxepane nucleotides, modified oxepane nucleotides, oligonucleotides, methods for making and uses for modulating gene expression. The oxepane nucleoside having formula I : wherein B is a heterocyclic base, R0 is selected from the group consisting of hydrogen and alkyl having one to eight carbons, R1 through R10 are as described in the descriptions and mirror image enantiomers thereof.

Description

OXEPANE NUCLEOSIDES AND OLIGONUCLEOTIDES, USES THEREOF AND METHODS OF MAKING THE SAME

Field of the Invention

The invention relates generally to oxepane nucleosides, and oligonucleotides containing at least one oxepane-modified nucleotide.

Background of the Invention

Nucleosides and chemotherapy. The discovery of nucleosides with antiviral and anticancer activity generally relies on the rational approach by which they are designed to act through (a) initial conversion to their 5' -triphosphate derivatives and inhibition of nucleotide polymerase through chain termination of the growing viral DNA or RNA chain, (b) inhibition of polymerase through mechanisms other than chain termination, (c) incorporation into the viral genome, thereby disrupting expression of genetic information or (d) inhibition of a metabolic pathway necessary for viral replication [Frontiers in Nucleosides and Nucleic Acids,- R. F. Schinazi and D. C. Liotta (Editors); IHL Press, 2004]. Despite significant advances in antiviral therapies, viral infections [e.g., West Nile virus, hepatitis C virus (HCV) , influenza virus] continue to be a serious threat in North America, with only a few potent and selective antiviral drugs reported to date. Chemistry of nucleosides in the area of anti-RNA-virus drug development has also been limited. The important targets here are hepatitis C virus and influenza viruses which replicate without the involvement of DNA. Nearly 170 million individuals are infected with HCV, and currently, there is no effective treatment for this infection [ Lauer, G. M. and Walker, B. D. (2001) Hepatitis C Virus Infection, N. Engl. J. Med., 345, 41-52.]. Acquired immunodeficiency syndrome (AIDS) , caused by the human immunodeficiency virus (HIV) , has become one of the most lethal chronic diseases for which no cure has yet been identified. Of the numerous lead compounds studied, only those that specifically target HIV-I reverse transcriptase or the HIV-I protease enzyme, and, more recently, the cell entry process, have been approved for HIV therapy. The common nucleoside reverse transcriptase inhibitors (NRTIs) e.g., the chain terminators AZT, 3TC, and d4T, and the non-nucleoside reverse transcriptase inhibitors (NNRTIs) e.g., nevirapine, efavirenz, and delavirdine effectively block viral DNA replication and slow the onset and progression of AIDS [(a) Mitsuya, H. et al . ,

(1990) Molecular targets for AIDS therapy, Science 249, 1533- 1544; (b) Kohlstaedt, L.A. et al . (1992) Crystal structure at

3.5 A resolution of HIV-I reverse transcriptase complexed with an inhibitor, Science, 256, 1783-1790; (c) Merluzzi, V.J. (1990)

Inhibition of HIV-I replication by a nonnucleoside reverse transcriptase inhibitor, Science, 250, 1411-1413].

Despite the tremendous success associated with antiretroviral combination therapy, which may also include inhibitors of the viral protease, the development of resistance cannot be prevented and accounts for a major cause of treatment failure. Moreover, studies have suggested that a significant number of newly infected individuals in Europe and North America harbor resistant variants of the virus. The prevalence and transmission of drug resistant variants is expected to increase, due to the extensive use of NRTIs, NNRTIs and protease inhibitors, which is an important factor that contributes to the limitations of treatment options for millions of infected individuals. Moreover, HIV has been classified in two subtypes: HIV-I and HIV-2. Most of the patients in North America and Europe are infected with HIV-I, while infection with HIV-2 and different variants of the HIV-I subtype, can be dominant in certain regions in Africa [Wainberg, M.A. (2004) HIV-I subtype distribution and the problem of drug resistance, AIDS, 18, S63- S68.] . The problem is that HIV-2 is nearly resistant to all known NNRTIs, which is an additional factor that can severely compromise and restrict current treatment strategies. Vaccination against HIV-I is still a long-range goal. Thus, the development of novel antiretroviral agents (such as novel nucleoside analogs) with potency against resistant HIV variants is of highest priority.

Oligonucleotide-based therapeutics. Oligonucleotide-based therapeutics have enormous potential for targeted therapy of cancer as well as inflammatory and infectious disease, exhibiting greater specificity and less toxicity than conventional chemotherapeutic drugs. The so-called "antisense" (AON) and "small interfering RNA" (siRNA) are the most prominent members of this class of agents. AONs and siRNAs can bind to a specific sequence of an mRNA target through base-pairing interactions, thereby interfering with expression of the protein encoded by the mRNA. Recently, it was determined that micro RNAs (miRNA) can also regulate expression of a large number of genes in plants and humans. This observation has led to the development of AONs that target miRNA [Meister, G. et al . (2004) Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing, RNA 10, 544-50; J. Krϋtzfeldt et al . , (2005) Nature 438, 685-689; Essau C. et al . (2004) Jourmal of Biological Chemistry 279, 52361-52365; Weiler, J. et al . 2006 Gene Therapy 13: 496-502] . Scientists are hoping to use AONs and siRNAs to design new, more effective, drugs to inhibit gene expression and production of abnormal levels of cell proteins. [Stull, R.A. and Szoka, F. C. (1995) Pharmaceutical Research, 12, 465-483; Uhlmann E. and Peyman, A. (1990) Chemical Reviews, 90, 544-584.; Mittal, V. (2004) Nature Rev., 5, 355-365]. Nucleic acid "aptamers" are a recent addition to the large number of nucleic acid-derived molecules being investigated as potential therapeutics. AONs and siRNAs are designed to target a specific mRNA, whereas nucleic acid "aptamers" (from the Latin word "aptus", meaning "to fit") exert their effect by binding to a specific target protein thus blocking such protein from further function [Nimjee, S. M. et al. (2005) Annu. Rev. Med. 56, 555- 583] .

AONs have been in clinical development for over a decade, and used in academic and commercial settings for validating biological function for over two decades. Initial clinical success for antisense appeared in the anti-infective areas (e.g. Vitravene™ for the treatment of CMV retinitis) . Whereas this particular drug has not proven to be a huge commercial success, it paved the way for a resurgent interest in oligonucleotide chemistries from both pharmaceutical companies and the biotechnology sector. The majority of clinical activity today with improved "second generation" chemistries is in the area of oncology. It is generally expected that second-generation antisense chemistries to treat inflammatory and autoimmune diseases will be the next wave of therapeutic agents, and signs of this are clearly evident in preclinical and several advanced clinical programs. However, the fact remains that the number of approved nucleic acid based drugs is minimal up to now.

More recently, RNA interference (RNAi) has emerged as an exciting potential alternative to the more classical antisense technology. There are several reports describing the utility of this method for silencing genes in living organisms, ranging from yeast to mammals. However, the majority of this work has been carried out with unmodified RNA in cell culture systems, which do not reflect the in vivo setting required for therapeutic applications of siRNAs. Thus, to obtain clinically useful molecules, it is desirable for antisense and siRNA molecules to have enhanced nuclease stability, as well as enhanced affinity for, and hybridization to, complementary RNA, since these physical attributes are beneficial if the molecules are to be viable as drug candidates . In addition, in the absence of a delivery vehicle, these molecules also need to be able to cross cell membranes and then hybridize with their intended RNA target. Also, RNA tertiary structure is a further factor which can affect the ability of antisense oligonucleotides and siRNA to hybridize with their RNA target. It is furthermore undesirable for either type of molecule to exert non-sequence- specific binding. Therefore, there is a need for improved oligonucleotide-based approaches. Further advances in this area will be achieved through improved chemistries that exhibit better efficacy and higher safety profiles, and are suitable to treat a wider variety of diseases.

To date, the largest expansion of the sugar moiety in DNA is that reported for six-membered ring hexopyranosyl nucleic acids and their assembly into (4'→6') linked oligo (2 ' , 3 ' -dideoxy-/?-D- glucopyranosyl) nucleotides (i.e. "homo-DNA" ). [Eschenmoser, A. (1999) Chemical Etiology of Nucleic Acid Structure, Science 284, 2118-2124] This architecture exhibits much stronger Watson-Crick base pairing than DNA, but it does not hybridize with native DNA or RNA strands. Since then, unsaturated 6-membered ring nucleic acids (e.g. cyclohexene NA) have been developed to promote binding to the natural systems in hybrid conformations that mimic the native duplexes [Wang, J. et al . Cyclohexene Nucleic

Acids (CeNA) : Serum Stable Oligonucleotides that Activate RNase

H and Increase Duplex Stability with RNA, J. Am. Chem. Soc.

(2000), 122, 8595-8602]. Alternatively, contracting the carbohydrate moiety to a four carbon skeleton, as in the αr-L- threofuranosyl sugar, maintains a furanose half-chair sugar pucker conformation similar to native systems, allowing α-L- threofuranosyl nucleic acids, TNAs, to pair with its complement as well as to single stranded, ssDNA and ssRNA [Schoning, K. -U. et al . (2000), Chemical Etiology of Nucleic Acid Structure: The α-Threofuranosyl- (3 ' ->2' ) Oligonucleotide System, Science 290, 1347-1351] .

Summary of the Invention

This invention relates to nucleosides and oligonucleotides comprised of a 7-membered heptose (oxepane) ring structure. The oxepane nucleosides and oligonucleotides of the present invention are capable of adopting a conformation that permits efficient pairing interactions (H-bonding and base stacking) for RNase H induction upon binding to complementary RNA target . Furthermore, siRNA duplexes incorporating the oxepane modification are shown to be capable of targeting (and silencing) cellular RNA through the RNA interference (RNAi) pathway. Oxepane thymine and adenine containing oiligonucleotides, in particular, were also found to be resistant to the nucleases present in fetal bovine serum (FBS) after 24 h incubation at 37°C.

According to one broad aspect of the invention, herein is provided, an oxepane nucleoside of formula (I) :

wherein ,

B is a heterocyclic base;

RO is selected from the group consisting of hydrogen and alkyl having 1 to 8 carbons; and

Rl through RlO are independently selected from the group consisting of a hydrogen, azido, amino, allyl, carboxyl, ester, halogen (fluorine, chlorine, bromine, and iodine) , hydroxyl, nitrile, sulfhydryl, alkyl, hydroxylmethy1 , alkylhalide, alkoxy, alkoxyalkyl, alkylsulfhydryl, allyl, propargyl, ethynyl, and ethenyl;

wherein any two of R₀ through Ri₀ may be covalently bonded to adjacent or non-adjacent ring carbons l'-6' to form a bicyclic structure; the covalently bonded group selected from the group consisting of -0-, -CH₂-, -CH₂CH₂-, -CH₂CH₂CH₂-, -OCH₂-, -OCH₂CH₂- , -CH₂OCH₂- ,-NH-, -NHCH₂-, -NHCH₂CH₂-, -CH₂NHCH₂-, -S-, -SCH₂CH₂-, and -SCH₂CH₂CH₂-,- and mirror image enantiomers thereof.

In one embodiment B is capable of base pairing and is preferably- selected from the group consisting of thymine, uracil, cytosine, adenine, guanine, inosine, 5-methylcytosine, 2-thiothymine, 4- thiothymine, 7-deazaadenine, 9-deazaadenine, 3-deazaadenine, 7- deazaguanine, 9-deazaguanine, 6-thioguanine, isoguanine, 2,6- diaminopurine, hypoxanthine , and 6-thiohypoxanthine .

In another embodiment, B is selected from the group consisting of 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, isocytosine, N⁴-methylcytosine, 5-iodouracil, 5-fluorouracil, 4- thiouracil, 2-thiouracil, (E) -5- (2-bromovinyl) uracil, N⁶- methyladenine, 2-chloroadenine, 2-fluoroadenine, 2- chloroadenine, N6-cyclopropyl-2, 6-diaminopurine, nicotinamide, 2-aminopurine, 1,2, 4-triazole-3-carboxamide.

According to another aspect of the invention, there is provided an oxepane nucleotide comprising the above oxepane nucleoside.

According to another aspect of the invention, there is provided an oligonucleotide comprising a plurality of nucleotides covalently bonded through a phosphorus containing moiety, wherein at least one nucleotide is an oxepane nucleotide. Preferably, the phosphorus containing moiety is selected from the group consisting of phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, H- phosphonate or phosphoramidate internucleoside linkages.

According to another aspect of the invention, there is provided a double stranded oligonucleotide comprising at least one oxepane nucleotide. The double stranded oligonucleotide may or may not have overhangs at the 3'-end(s) .

According to another aspect of the invention, there is provided a method for increasing at least one of therapeutic efficacy, nuclease stability, and/or selectivity of binding of an oligonucleotide, the method comprising inserting or replacing at least one nucleotide of the oligonucleotide with an oxepane nucleotide .

According to another aspect of the invention, there is provided a method of selectively modulating gene expression by administering an oligonucleotide or double stranded oligonucleotide having at least one oxepane nucleotide.

According to another aspect of the invention, there is provided pharmaceutical compositions comprising any of the foregoing with a pharmaceutically acceptable carrier.

According to another aspect of the invention, there is provided uses corresponding to the methods of the present invention.

According to another aspect of the invention, there is provided use of the oligonucleotides of the present invention for the preparation of a medicament for modulating gene expression.

According to another aspect of the invention, there is provided methods for making the oxepane nucleosides and oxepane nucleotides and oligonucleotides containing the same.

Brief Descriptions of the Drawings

The invention will now be described in greater detail having regard to the appended Figures in which:

Figure 1 indicates solid phase oligonucleotide synthesis of OT₁₅ and oA_i5 oligonucleotides. (A) is a schematic of the base unit structure of an oT (left) and an oA (right) nucleoside. (B) shows a unylinker derivatized 500A support. (C) is an image of an electrophoretic gel characterization of OT₁₅, oA₁₅, dT₁₅, dA₁₅, rU₁₅ and rA₁₅ oligonucleotides. A 0.5 μmol scale provided efficient coupling (overall yields up to 80%) with amidite concentration as low as 0.04-0.05M and extended coupling times of coupling times of 30min. (D) is a graphical representation of HPLC and mass spectrometry characterization of the synthesized oxepane nucleosides. Figure 2 shows: (A) melting temperature curves of oxepane oT₁₅: oA₁₅ duplex and the corresponding native duplex dT₁₅:dA₁₅; and (B) job plots at 5°C to determine the stoichiometry of interaction (1:1 for both systems, suggesting formation of a duplex structure under these conditions) .

Figure 3 shows the effect of temperature on the circular dichroism spectrum of hybrid duplex rA₁₅:dT₁₅ (buffer: 5mM Na₂HPO₄, 14OmM KCl and ImM MgCl₂).

Figure 4 shows the effect of temperature on the circular dichroism spectrum of duplex rA₁₅:oT₁₅. A plot of the peak at around 250 nm allows for the calculation of the melting temperature of this hybrid duplex (ca. 13 °C) (buffer: 5mM Na₂HPO₄, 14OmM KCl and ImM MgCl₂) .

Figure 5 shows the results of: (A) a nuclease assay to assess the stability of OT₁₅ versus dT₁₅ against exonucleases and endonucleases present in 10% fetal bovine serum; and (B) polyacrylamide gel electrophoresis analysis of intact oligonucleotides .

Figure 6 shows the results of ribonuclease H degradaton of three 15-bp oligonucleotide hybrid duplexes. A 15-nt 5' -³²P-labeled target RNA (rA₁₅) was pre-incubated with complementary 15-nt dτ₁₅, oT₁₅, and rU₁₅, and then added to reaction assays containing E. coli RNase H at the indicated temperature. The data (A and B) show that both dT₁₅ and OT₁₅ elicit RNase H activity.

Figure 7 shows the activity of oxepane-modified siRNAs targeting the luciferase firefly mRNA (duplex sequences are shown at the right) . Concentrations of duplexes were varied from 8OnM to 0.0006 nM. Oxepane nucleotides are highlighted (colored, underlined fonts) .

Figure 8 is a scheme of the synthesis of the silyl group protected and acetylated cyclopropanated sugar (E5) for use in synthesis of oxepane modified nucleosides. Conditions and reagents are as follows: i. 0.15 M NaOMe/MeOH, 22°C, 3 h, 95%; ii. t-Bu₂Si(OTf)₂, DMF, pyr, -40⁰C, 1 h, 80%; iii. 1 M ZnEt₂/hexanes, CH₂I₂, Et₂O, 0⁰C, 5 h, 81%; and iv. Ac₂O, pyr, 22°C, 1 h, 99%. Structures are as follows: 1- tri-O-acetyl D- glucal; 2: D-glucal; 3_^ ^: 4 ' , 6' -O-bis-siloxane protected sugar,- 4_: cyclopropanated sugar; and ^: silyl group protected and acetylated cyclopropanated sugar.

Figure 9 is a scheme of the synthesis of oxepane modified nucleosides for use in oligonucleotide synthesis reactions. A silyl group protected and acetylated cyclopropanated sugar is reacted with a nucleobase (B) . Conditions and reagents are as follows: i. persilylated base, TMSOTf, MeCN, reflux, 12-24 h, Thy (a): 40% and AdeNBz (b) : 45%; ii. 1 M TBAF/THF, 0⁰C, 1 h, Thy (8) : 90% and AdeNBz (£) : 61%; iii. 1 atm H₂, Pd/C, MeOH, 22°C, 4 h, Thy (1£) : 70% and AdeNBz (11) : 99%; iv. MMT-Cl, pyridine, 22°C, 3 h, Thy (12) : 66% and AdeNBz (13) : 50%; and v. Cl-P(OCEt)N(I-Pr)₂, Et-N(I-Pr)₂, THF, 22°C, 2 h, Thy (14): 80%, and AdeNBz (15): 90%.

Figure 10 is a table showing the selectivity of the glycosylation reaction of the silyl group protected and acetylated cyclopropanated sugar, !>.

Figure 11 shows alternate schemes for synthesizing second generation modified oxepane nucleosides 16^ VJ_, 18_^, and 19.

Figure 12 is a table showing the Tm values (⁰C) of complexes given as a comparison of the UV thermal melt, Tm for pairing and cross-pairing of oxepane, OT₁₅ and OA₁₅ oligonucleotides and the control DNA (dT₁₅ and dA₁₅) and RNA (rU₁₅ and rA₁₅) sequences.

^♦Represents rough Tm due to early and/or broad transition curve.

Comparison of the Tm per each modified oxepane insert (oT and oA) , within an RNA duplex is indicated. All sequences contained a duplex concentration of 3.04 μM in sodium phosphate buffer,

140 mM KCl, 1 mM MgCl₂, 5 mM Na₂HPO₄, Detailed Description

In the following description, numerous specific details are set forth to provide a thorough understanding of the invention. However, a person skilled in the art will understand, given the context, that circumstances exist in which the invention may be practiced without specific preferred features. In the following description reference is made to certain terms of the art.

"Nucleosides" are individual units consisting of a heterocyclic base covalently bonded to a cyclic sugar. In some embodiments, the base is any heterocyclic base capable of base pairing with other heterocyclic bases, and includes any one of the natively found purine and pyrimidine bases, adenine (A) , thymine (T) , cytosine (C) , guanine (G) and uracil (U) , but also any modified or analogous forms thereof. Examples of non-naturally occurring bases that are capable of forming base-pairing relationships include, but are not limited to, aza and deaza pyrimidine analogues, aza and deaza purine analogues, and other heterocyclic base analogues, wherein one or more of the ring atoms and/or functional groups of the purine and pyrimidine rings have been substituted by heteroatoms, e.g., carbon, fluorine, nitrogen, oxygen, sulfur, and the like. Preferably, such bases include, but are not limited to, inosine, 5- methylcytosine, 2-thiothymine, 4-thiothymine, 7-deazaadenine, 9- deazaadenine, 3-deazaadenine, 7-deazaguanine, 9-deazaguanine, 6- thioguanine, isoguanine, 2, 6-diaminopurine, hypoxanthine, and 6-thiohypoxanthine . Bases may also include, but are not limited to, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, isocytosine, N⁴-methylcytosine, 5-iodouracil, 5-fluorouracil, 4- thiouracil, 2-thiouracil, (E) -5- (2-bromovinyl) uracil, N⁶- methyladenine, 2-chloroadenine, 2-fluoroadenine, 2- chloroadenine, N6-cyclopropyl-2, 6-diaminopurine, nicotinamide, 2-aminopurine, 1, 2, 4-triazole-3-carboxamide . The sugar is traditionally a naturally occurring 5-carbon sugar such as 2- deoxyribose, or ribose but in embodiments of the present invention is oxepane (a seven-carbon sugar) and derivatives thereof .

"Nucleotides" are nucleoside units further having a phosphorus moiety covalently bonded to the sugar moiety of the nucleoside, preferably at either the 3' or the 5' position of the sugar.

"Modified nucleoside" refers to a nucleotide that differs from a naturally occurring nucleotide in some modification and can be made by chemical modification of the sugar unit or nucleoside base .

"Modified nucleotide" refers to a nucleotide that differs from a naturally occurring nucleotide in some modification and can be made by chemical modification of the phosphate backbone, sugar unit or nucleoside base.

"Oxepane nucleoside" refers to that specific modified nucleoside in which the furanose sugar moiety of naturally occurring nucleosides has been replaced by a seven-membered ring oxacycloheptane, C₆Hi₂O, also called oxepane (P. Luger et al., Acta Cryst. (1991) . C47, 102-106) .

"Oxepane nucleotides" are oxepane nucleoside units further having a phosphorus moiety covalently bonded to the sugar moiety of the nucleoside.

"Modified oxepane nucleoside" includes, but is not limited to an oxepane nucleoside containing functional groups (substituents) on the oxacycloheptane ring structure. For example, the oxepane modified nucleotide may comprise substituents selected from the group consisting of (but not limited to) a double bond, azido, amino, allyl, halogen, hydroxyl, sulfhydryl (SH), alkyl and functionalized alkyl groups. These functional groups or substituents may be oriented above (beta) or below (alpha) the plane defined by oxacycloheptane ring. The above-mentioned oxepane nucleotide comprises heterocyclic bases selected from the group consisting of adenine, cytosine, guanine, thymine, uracil, and other heterocycles such as inosine, 5- methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5- iodocytosine, isocytosine, N⁴-methylcytosine, 5-iodouracil, 5- fluorouracil, 4-thiouracil, 2-thiouracil, (E) -5- (2- bromovinyl) uracil, 2-thiothymine, 4-thiothymine, 7-deazaadenine, 9-deazaadenine, N⁶-methyladenine, 2-chloroadenine, 2- fluoroadenine, 2-chloroadenine, isoguanine, 3-deazaadenine, 7- deazaguanine, 9-deazaguanine, 6-thioguanine, nicotinamide, 2- aminopurine, 2 , 6-diaminopurine, Nδ-cyclopropyl-2, 6- diaminopurine, hypoxanthine , 6-thiohypoxanthine, 1, 2, 4-triazole- 3-carboxamide.

Example of a modified oxepane nucleoside is, but is not limited tθ:

In the above example, the methoxyethoxy and hydroxyl substituents have the alpha ("down") configuration, whereas the heterocyclic base, hydroxylmethyl and ethyl groups are oriented in the beta ("up") configuration. It would be understood by a person skilled in the art that substituents may be either in the alpha or beta configuration. It would also be understood by a person skilled in the art that the base moiety ("B") may be located at other positions of the sugar ring (i.e. in addition of being located at Cl', as shown, it may be placed at C2', C3 ' , C4 ' and C5 ' ) .

The oxepane nucleoside may also exist in two stereoisomeric forms, for example a pair of enantiomers: mirror

"Modified oxepane nucleotides" are modified oxepane nucleoside units further having a phosphorus moiety covalently bonded to the sugar moiety of the nucleoside.

"Oligonucleotides", or "oligomers", are polymers of at least two nucleoside units, wherein each of the individual nucleoside units is covalently linked to at least one other nucleoside unit through a single phosphorus moiety. In the case of naturally occurring oligonucleotides, the covalent linkage between nucleoside units is a phosphodiester bond. Oligonucleotides as defined herein are comprised of about 1 to about 100 nucleotides, more preferably from 1 to 80 nucleotides, and even more preferably from about 10 to about 50 nucleotides. Nevertheless, the term "oligonucleotide" as used herein includes, but is not limited to, oligonucleotides that are modified with respect to any one or more of the following: (1) the phosphodiester bond between nucleoside units, (2) the individual nucleoside units themselves and/or (3) the ribose, or sugar, moiety of the nucleoside units. Such modified oligonucleotides are best described as being functionally interchangeable with, yet structurally different from, natural oligonucleotides. Representative modifications include phosphorothioate, phosphorodithioate, methylphosphonate, H- phosphonate, phosphotriester or phosphoramidate internucleoside linkages in place of phosphodiester internucleoside linkages; these phosphates and phosphate modifications can be present at any of the positions of the oxepane ring,- deaza or aza purines and pyrimidines in place of natural purine and pyrimidine bases, pyrimidine bases having substituent groups at the 5 or 6 position; purine bases having altered substituent groups at the 2, 6 or 8 positions or 7 position as 7-deazapurines; sugar units containing 5, 6 and 7-membered ring structures. In addition, modification can be made wherein nucleoside units are joined through groups that substitute for the internucleoside phosphate or sugar phosphate linkages.

"capable of hybridization" means the ability to hybridize under the following conditions: 100 mM Tris HCl pH: 7.5, 100 mM KCl, 50 mM MgCl₂, 0.5 mM EDTA, 0.5 mM DTT at 4⁰C.

In one aspect, there is described an oxepane nucleoside of formula (I) :

wherein ,

B is a heterocyclic base,-

R₀ is selected from the group consisting of hydrogen and alkyl having 1 to 8 carbons; and

R₁ through R₁₀ are independently selected from the group consisting of a hydrogen, azido, amino, allyl, carboxyl, ester, halogen (fluorine, chlorine, bromine, and iodine), hydroxyl, nitrile, sulfhydryl, alkyl, hydroxylmethyl , alkylhalide, alkoxy, alkoxyalkyl, alkylsulfhydryl, allyl, propargyl, ethynyl, and ethenyl ;

wherein any two of R₀ through R₁₀ may be covalently bonded to adjacent or non-adjacent carbons l'-6' to form a bicyclic structure; the covalently bonded group selected from the group consisting of -0- , -CH₂-, -CH₂CH₂-, -CH₂CH₂CH₂-, -OCH₂-, -OCH₂CH₂- ,-CH₂OCH₂-, -NH-, -NHCH₂-, -NHCH₂CH₂-, -CH₂NHCH₂-, -S-, -SCH₂CH₂-, and -SCH₂CH₂CH₂-; and mirror image enantiomers thereof. Preferably, R₀ is hydrogen.

In some embodiments, B is capable of base pairing and is preferably selected from the group consisting of thymine, uracil, cytosine, adenine, guanine, inosine, 5-methylcytosine, 2-thiothymine, 4-thiothymine, 7-deazaadenine, 9-deazaadenine, 3- deazaadenine, 7-deazaguanine, 9-deazaguanine, 6-thioguanine, isoguanine, 2 , 6-diaminopurine, hypoxanthine, and 6- thiohypoxanthine .

In some embodiments, B is selected from the group consisting of 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, isocytosine, N⁴-methylcytosine, 5-iodouracil, 5-fluorouracil, 4-thiouracil, 2-thiouracil, (E) -5- (2-bromovinyl) uracil, N⁶-methyladenine, 2- chloroadenine, 2-fluoroadenine, 2-chloroadenine, N6-cyclopropyl- 2 , 6-diaminopurine, nicotinamide, 2-aminopurine, 1, 2, 4-triazole- 3 -carboxamide .

In some embodiments the alkyl group is selected from the group consisting of methyl, ethyl, propyl, butyl, and functionalized alkyl groups thereof, preferably selected from the group consisting of methylamino, dimethylamino, ethylamino, diethylamino, propylamino and butylamino groups.

In some embodiments, the alkoxy group is selected from the group consisting of methoxy, ethoxy, propoxy and functionalized alkoxy groups thereof, preferably selected from the group consisting of -0 (CH₂) _q-R, where q=2-4 and R is -NH₂, -OCH₃, or -OCH₂CH₃.

In some embodiments, the alkoxyalkyl group is selected from the group consisting of methoxyethyl, and ethoxyethyl .

In one embodiment the oxepane nucleoside has the structure of formula (II) :

II. In another aspect, there is provided an oxepane nucleotide comprising the oxepane nucleoside described herein.

In another aspect there is provided an oligonucleotide comprising a plurality of nucleotides covalently bonded through a phosphorus containing moiety, wherein at least one nucleotide is an oxepane nucleotide. Preferably, the phosphorus containing moiety is selected from the group consisting of phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, and phosphoramidate internucleoside linkages. The oligonucleotide is preferably 15-50 nucleotides in length.

In some embodiments, the oligonucleotide is capable of hybridizing to and inducing RNase H-mediated cleavage of an RNA strand.

In some embodiments the oligonucleotide is 15-80 nucleotides in length and exhibits self-complementarity thereby being capable of adopting a hairpin duplex structure.

In another aspect, there is provided a double stranded oligonucleotide comprising at least one oligonucleotide having an oxepane nucleotide. In some embodiments, one or both strands have overhangs from 1-5 nucleotides on the 3' -end. In other embodiments neither strand has an overhang.

In another aspect, there is provided a method for increasing at least one of therapeutic efficacy, nuclease stability, and/or selectivity of binding of an oligonucleotide, the method comprising inserting or replacing at least one nucleotide of the oligonucleotide with an oxepane nucleotide.

In another aspect, there is provided a method of selectively modulating gene expression by administering an oligonucleotide and/or double stranded oligonucleotide described herein.

In another aspect, there is provided a method of selectively modulating gene expression by administering a double stranded oligonucleotide described herein. In another aspect, there is provided a pharmaceutical composition comprising an oligonucleotide described herein and a pharmaceutically acceptable carrier.

In another aspect, there is provided a pharmaceutical composition comprising a double stranded oligonucleotide described herein and a pharmaceutically acceptable carrier.

In another aspect, there is provided a pharmaceutical composition comprising a nucleoside described herein a pharmaceutically acceptable carrier.

In another aspect, there is provided a pharmaceutical composition comprising a nucleotide described herein and a pharmaceutically acceptable carrier.

In another aspect, there is provided use of an oligonucleotide and/or double stranded oligonucleotide described herein for modulating gene expression.

In another aspect, there is provided use of an oligonucleotide and/or double stranded oligonucleotide described herein in the preparation of a medicament for modulating gene expression.

In another aspect, there is provided use of an oligonucleotide and/or double stranded oligonucleotide described herein a compound selected from the group consisting of compounds of formulas (III-X) :

III

10

wherein B is selected from the group consisting of thymine, uracil, cytosine, adenine, guanine, inosine, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, isocytosine, N⁴-methylcytosine, 5-iodouracil, 5-fluorouracil, 4-thiouracil, 2-thiouracil, (E) -5- (2-bromovinyl) uracil, 2-thiothymine, 4- thiothymine, 7-deazaadenine, 9-deazaadenine, N⁶-methyladenine, 2-chloroadenine, 2-fluoroadenine , 2-chloroadenine, isoguanine, 3-deazaadenine, 7-deazaguanine, 9-deazaguanine, 6-thioguanine, nicotinamide, 2-aminopurine, 2, 6-diaminopurine, N6-cyclopropyl- 2, 6-diaminopurine, hypoxanthine , 6-thiohypoxanthine, and 1,2,4- triazole-3-carboxamide,

and mirror image enantiomers thereof.

In another aspect, there is provided a method of preparing an oxepane nucleoside comprising the step of reducing the double bond of an oxepine nucleoside with a reducing agent. Preferably, the double bond is between the 3' carbon and the 4..' carbon and the reducing occurs in the presence of a palladium metal pre- catalyst such as palladium/charcoal.

In some embodiments, the oxepine nucleoside is produced by deprotecting a protected oxepine nucleoside, preferably the protected oxepine nucleoside is protected at the 5' carbon and the 6' carbon by a silyl group. More preferably, the silyl group is a siloxane group.

A person skilled in the art would understand that other protecting groups could be chosen and are available, including, but not limited to Benzylidene acetal, acetyl (Ac) , benzoyl (Bz) , benzyl (Bn) , and other silyl groups such as tert- butyldimethysilyl (TBDMSi) , tert-butyldiphenylsilyl (TBDPSi) and as further disclosed in available references, such as "Protecting groups", by Von P.J. Kocienski (Thieme, Stuggart) , 1994.

In some embodiments, the protected oxepine nucleoside is produced by glycolsyating a nucleobase with a protected cyclopropanated hexopyranose .

Preferably, the nucleobase is selected from the group consisting of thymine, uracil, cytosine, adenine, guanine, inosine, 5- methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5- iodocytosine, isocytosine, N⁴-methylcytosine, 5-iodouracil, 5- fluorouracil, 4-thiouracil, 2-thiouracil, (E) -5- (2- bromovinyl) uracil, 2-thiothymine, 4-thiothymine, 7-deazaadenine, 9-deazaadenine, N⁶-methyladenine, 2-chloroadenine, 2- fluoroadenine, 2-chloroadenine, isoguanine, 3-deazaadenine, 7- deazaguanine, 9-deazaguanine, 6-thioguanine, nicotinamide, 2- aminopurine, 2 , 6-diaminopurine, N6-cyclopropyl-2, 6- diaminopurine, hypoxanthine, 6-thiohypoxanthine, and 1,2,4- triazole-3-carboxamide .

The nucleobase may be protected with a silyl group where appropriate. Typically, nucleobases requiring protection are those containing exocyclic amino groups (guanine, adenine and cytosine and derivatives of these that retain the amino groups) . Typical protecting groups for these nucleobases include acetyl (Ac) , benzoyl (Bz) , isobutyryl (i-Bu) , dimethylformamidine (dmf) , and levulynyl (Lv) groups. A person skilled in the art would understand the nucleobases requiring protection and the corresponding protecting groups used, for example as described in "Protection of Nucleosides for Oligonucleotide Synthesis", Current Protocols in Nucleic Acid Chemistry, Chapter 2, by Serge L. Beaucage, 2005 John Wiley & Sons, Inc.

In some embodiments, the cyclopropanated hexopyranose is protected via silylation and is acetylated. In some embodiments, the cyclopropanated hexopyranose is acetylated at the 3' position. Preferably, the protected cyclopropanated hexopyranose is comprised of a sugar selected from the group consisting of a D-glucal and L-glucal . More preferably, the cyclopropanated hexopyranose is acetylated 5' , 6' -O-bis-siloxane-protected D- glucal .

In some embodiments, the protected cyclopropanated hexopyranose is produced by cyclopropanating a protected, unsaturated hexopyranose sugar. Preferably, the cyclopropanation of the protected sugar occurs under Simmons-Smith conditions.

In some embodiments, acetylation of the cyclopropanated hexopyranose proceeds as :

In some embodiments, cyclopropanation of the protected, unsaturated hexopyranose proceeds as :

A person skilled in the art would understand appropriate metal catalysts to be used in the above reaction [A. B. Charette et al . Angew. Chemie Int. Edition (2000) 112: 4713]. Preferably, organozinc compounds are used.

In some embodiments, the protected, unsaturated hexopyranose is produced by:

In some embodiments, glycosylation of the nucleobase proceeds as :

wherein B is a free or protected heterocyclic base.

In some embodiments, deprotection of the protected oxepine nucleoside proceeds as:

HO 3

HO In some embodiments, reduction of the oxepine nucleoside proceeds as :

H₂ source catalyst

In some embodiments, there is described protecting the oxepane nucleoside and phosphitylating the protected oxepane nucleoside to yield a protected oxepane nucleoside phosphoramidite derivative for use in oligonucleotide synthesis. Preferably, the oxepane nucleoside is protected at a hydroxyl group by tritylation. More preferably, the tritylation and phosphitylation proceed as:

Other common trityl groups which could be used to protect the oxepane nucleoside are monomethoxytrityl (MMT) and dimethoxytrityl (DMT) , with the latter being the more commonly used for oligo synthesis. Further, a person skilled in the art would understand that other protecting groups could be used to the replace the trityl group in the oxepane phosphoramidites, such as a levulinyl (Lv) group. Preferably, the methods of the invention proceed as outlined in the Schemes herein.

In another aspect, there is provided a phosphoramidite derivative comprising a nucleoside described herein.

In another aspect, there is provided use of an oxepane nucleoside described herein for synthesis of oligonucleotides, wherein the oxepane nucleoside is subjected to tritylation and phosphitylation prior to synthesis.

The methods described here provide unprecedented oxepane nucleosides and oligonucleotides. The total synthesis of the oxepane nucleosides and derivatives starts from commercially available tri-O-acetyl D-glucal (derived from D-glucose) (Scheme 1 through 3) . The starting material 1 is initially completely deacetylated (methanolic sodium methoxide) in quantitative yields to generate D-glucal 2, which upon solvent evaporation yields a white crystalline solid. D-glucal 2 can be regioselectively protected at -40⁰C for 1 hour with t-Bu₂Si (OTf) ₂ to yield the 4',6'-O-bis siloxane protected sugar 3 as a white crystalline product in excellent yield (Scheme 1) . The Simmons- Smith cyclopropanation conditions yields the cylcopropanated sugar 4 as its pure diastereomer in good yield. Acetylation of the allylic alcohol group to give 5 proceeds quantitatively prior to the Vorbrϋggen-like glycosylation reaction (Scheme 2) . Thymine or N6 benzoyladenine is initially silylated with 1,1,3,3,3- hexamethyldisilazane (HMDS) under reflux conditions and the protected sugar dissolved in anhydrous MeCN was transferred to the silylated base and stirred. 30 mol% of TMSOTf was added and the resulting mixture refluxed to yield a mixture of nucleoside anomers 6 and the diene bi-product 7. Coupling with Λ^-benzoyladenine proceeded more rapidly and more efficiently than with thymine (0.5 days vs 1 day reflux with a chemoselectivity of 40%: 30% for £a and T_, and 45%: 15% for 6b and T) at the expense of a poorer diastereoselectivity (2:1 β:a for ^-benzoyl adenine and 10:1 β:a for thymine), (Table 2). The isolated /?-anomers of the oxepine nucleosides jia and 6b were desilylated with 1 M TBAF in THF until TLC predicted complete reaction. The oxepine nucleosides were hydrogenated with palladium/charcoal, (Pd/C) and the oxepane nucleosides, 1Q_, (oxepane T) and ^l, (oxepane A) were tritylated and phosphitylated to the 7'-MMT 5' -phosphoroamidite derivatives, 14_^ and 15.

All nucleoside products were analyzed and characterized by 1-2D ¹H, ¹³C NMR and ESI MS. For example, the absolute stereochemistry at the anomeric position was confirmed by NOESY experiments which indicated a strong through space coupling cross-peak for the anomeric 1' and 6' protons for the /?-anomers of the oxepine nucleosides 6ia and ^b. The reduction of the oxepine double bond was monitored by ¹H NMR, which indicated complete conversion to the saturated oxepane nucleosides, 10, (oT) and ^l, (oA) by the disappearance of the oxepine vinylic hydrogen protons, 3' and 4' at approximately δ: 6.0. These were finally converted to the tritylated nucleoside phosphoramidite diastereomers, 14_^ and 3J>, and analyzed for purity by ³¹P (¹H decoupled) NMR (14: δ 149.5 and 149.0 and 15: δ 148.4 and 147.9) and their structures confirmed by ¹H NMR and by molecular weight with ESI mass spectrometry (i.e. 14_: C₄₁H₅₁N₄O₇PNa: 765.9, found 765.2 and 15: C₄₈H₅₄N₇O₆PNa 878.9, found 878.3). Full characterization data are provided in the examples later described.

The functionalization of the oxepine double bond in oT* (6a or JJ) provided a variety of modified ONA derivatives (Scheme 3) . For example, epoxidation of ^a with m-chloroperbenzoic acid (inCPBA) was successively achieved to yield target compound 16, in 50% yield. Modest diastereoselectivity was observed, favoring the α-epoxy nucleoside to yield an inseparable mixture of 2 a-.lβ after 24 hours reaction at 40⁰C. The structure of the α-cis oxirane nucleoside, ^6_ was confirmed by the NOESY cross- peak assignments and the strong syn coupling between H3 ' and H4' (³J₃M- : 10.6 Hz) . The dihydroxylation reaction of ^a with catalytic (7 mol %) osmium tetroxide (OsO₄) in the presence of W-morpholine N-oxide, (NMO) as re-oxidant was found to generate the diol, 17.' i-^{n a 50}% yield. The reaction was completed after 5 hours generating the diastereomers, VT_ as a 1:1 inseparable mixture of a:β cis hydroxylated isomers.

The cyclopropanation reaction with £a following Furukawa's methods yielded the cyclopropanated oxepane nucleoside, 3J5, in 30% yield. The reaction yielded selectively, the α-cis cycloproponated oxepane nucleoside, IjJ. The structure was confirmed by assignment of the COSY and NOESY crosspeaks and the strong syn proton coupling constant ³J₃.^: 12 Hz.

The regioselective mono-hydroxylation reaction with £a was also attempted for the synthesis of target compound \S_. Thus, hydroboration with borane-THF (BH₃-THF) proceeded slowly, preferably using an overnight reaction for completion. This was followed by the base hydrolysis (H₂O, NaOH) and oxidation (H₂O₂) reactions for 2 hours which generated the product diastereomers (2.5:1 ratio; 67% total yield) in favor of the /?-cis adduct, 19. The /?-cis stereochemistry of 19 was established by NOESY cross- peaks and was rationalized by the steric influence of the neighboring 5'-silyl ether protecting group which prevented attack from the bottom face of the oxepine ring. This protecting group also favored a 10:1 regioselectivity by facilitating the delivery of BH₃ to the C4 ' (relative to the C3 ' ) position of the reagent, 6a.

The novel nucleoside derivatives described above contain a 7- membered heptose carbohydrate moiety. Each of these derivatives (in their completely deprotected form) represents potential nucleoside antivirals, or after elaboration into phosphoramidite derivatives, represent building blocks for oxapane oligonucleotides via solid-phase synthesis.

For example, the oxepane phosphoramidite derivatives 14. ^an<^ Ikϋ. can be readily assembled into oxepane (V) oligonucleotides, e.g., 0T₁₅ and OA₁₅, by conventional automated solid phase synthesis cycle. These derivatives were used as dilute 0.05 M solutions, in anhydrous CH₂Cl₂ for the more non-polar 14 (R_f: 2:1 Hex: EtOAc, 0.52), and in anhydrous MeCN for the more polar ]J5 (R_f: 2:1 Hex : EtOAc , 0.33). Oligonucleotide syntheses were conducted on a 0.5 μmol scale using extended amidite coupling times (30 min) and 5-ethylthiotetrazole, ETT, as the activator (0.25 M in MeCN) in order to ensure efficient coupling. The detritylation step was extended to 2.5 minutes for the complete removal of the MMT groups. All other control DNA and RNA sequences were synthesized using conventional procedures. Recoveries of OT₁₅ and OA₁₅ from the solid support were ca. 25 OD units, (25-40% yield) . Furthermore, the desired oligonucleotides constituted 65-70% of the crude material isolated after alkaline deprotection, indicating that the monomers coupled with 98-99% stepwise efficiency and were chemically resistant to the deprotection conditions (Figure 1) . Following purification by AE HPLC and/or denaturing PAGE conditions, the oligomers were desalted by size exclusion chromatography (Sephadex^® G-25) . The same procedures were followed for the synthesis of RNA strands incorporating oxepane nucleotide units. Table 1 lists the oligonucleotides prepared. The structures of the oligonucleotides were confirmed by MALDI-TOF mass spectrometry (e.g., OT₁₅, 4924 g/mol, found: 4927; oA_15> 5056 g/mol; found: 5080) .

The biochemical and physicochemical properties of the ONA strands were evaluated for the very first time (Figures 2-7) . The data obtained conclusively showed that ONA exhibit certain characteristics desirable for use as gene silencing agents. Like the native DNA or RNA, ONA were shown to form 1:1 complexes with themselves (i.e. oT₁₅:oA₁₅ duplex; Figure 2), and in the case of OT₁₅, with complementary target RNA as well (Figure 4; Table 1) . The oT₁₅/rA₁₅ hybrid duplex adopted a global helical conformation of the A-family, with more A-form than B-form character. This property is shared with the unmodified dT₁₅/rA₁₅ control hybrid, whose CD patterns matched very well those of the corresponding oT₁₅/rAi₅ duplex (compare CD spectra of Figures 3 and 4) . Furthermore, O¹T₁₅ displays significant resistance to 3 ' -exonucleases present in serum (Figure 5) and, surprisingly, was able to direct E. coli RNase H degradation of target RNA molecules (Figure 6) . Less hydrolysis occurs when compared with the native substrate which can be partly rationalized by the lower affinity of oT₁₅ for the RNA target (T_n, ~ 13°C) . This property acts to present a lower effective concentration of substrate duplex to the enzyme, thereby diminishing the overall rate of catalysis. Induction of RNase H activity is likely to have therapeutic value by enhancing the antisense effect relative to oligomers that are unable to activate this enzyme [Juliano, R. L. et al . (1999) Pharm. Res. 16, 494-502; Walder, R. Y., and Walder, J. A. (1988) Proc. Natl. Acad. Sci . U.S.A. 85, 5011-5015] . It should be emphasized that of the hundreds of modified oligonucleotides known, only a handful exhibit these combined properties [Mangos, M. and Damha, M.J. (2002) Current Topics in Medicinal Chemistry, 2: 1145-1169].

The present invention also affords short interfering RNA (siRNA) duplexes containing the oxepane modification. The thermal denaturation data (Table 1) indicated that oxepane modifications are tolerated in siRNA duplexes (ΔT_m -1 to -5 ⁰C) , provided that they are not introduced in the center of the helix, where destabilization in this particular duplex was more significant (Δ T_n, -11 ⁰C) . Of significance, these duplexes can enter the RNA interference (RNAi) pathway. More specifically, oxepane modified siRNA were shown to inhibit the expression of the enzyme luciferase, by targetting the mRNA encoded by the luciferase gene. Thus, the modified siRNAs 11-16 (Table 1) and the control siRNA duplex 10, were tested in the HeIa cell line that over-expresses the firefly luciferase protein. Assays were carried out following literature procedures [Dowler, T. (2006) Improvements in siRNA properties mediated by 2'-deoxy 2'-fluoro- /?-D-arabinonucleic acids (FANA) Nucl. Acids Res. 34, 1669- 1675.] . Samples were transfected at five different concentrations for 24 h and the cells harvested for determination of the protein firefly luciferase counts normalized against a scrambled siRNA control. As this control does not bind to the firefly mRNA, any effects on the firefly production due to the transfecting agent (lipofectamine 2000 from InVitrogen Inc.) will be taken care of. The data (Figure 7) shows that oxepane-modified siRNAs were found to be less active relative to the control sequence 10, however, certain sequences particularly those containing the oN substitution in the sense RNA strand (e.g. duplexes 11, 12 and 13) maintained RNAi activity up to the low nM concentrations (1-10 nM) . Modifications introduced in the antisense RNA strand (e.g., 14, 15 and 16) and at the 5' -end of the siRNA duplex (14) were also found to be less tolerated. However, this effect was found to be compensated to a certain extent by re-introducing oN modifications in the sense strand (e.g. 15 vs. 16) . It is remarkable that the most modified siRNA duplex (15) inhibits luciferase activity by 50% at a concentration of only 4 nM.

The examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein.

Example 1; Preparation of Starting Materials

4,6-0- (di-tert-butylsilanediyl) -D-glucal U). The starting material, 1, (8 g, 29.38 mmol) was vacuum dried overnight prior to initiating the reaction the following day. At ambient temperatures (22°C) and with N₂, MeOH (40 mL) was added to dissolve the starting material and the reaction was initiated with the addition of a freshly prepared solution of 2 M NaOMe in MeOH (0.5 mL, 0.13 M). The reaction was completed after 1.5 h, as was indicated by TLC, with R_f: (9: 1 CH₂Cl₂ : MeOH) 0.25. The crude reaction mixture was concentrated in-vacuo to a crude oil and purified by silica gel column chromatography. The crude was eluted with 9:1 v/v CH₂Cl₂ :MeOH and the purified product 2, was collected in yields of 4.30 g (99%) as a white crystalline solid after drying with high vacuum.

D-glucal, 2_, (4.30 g, 29.42 mmol) was flushed with a constant flow of N₂ and at -40⁰C was added anhydrous DMF (145 mL, 2.87 mol) and stirred to complete solution. The reaction was initiated with the slow (dropwise for 5 min) addition of t- Bu₂Si(OTf)₂, (15 mL, 40 mmol) and the reaction was completed after 45 min by confirmation with TLC, R_f : (5 : 1 Hex : EtOAc) 0.33. The crude reaction mixture was quenched with anhydrous pyr (3.6 mL, 44 mmol) and stirred for an additional 15 min. The crude product was extracted in Et₂O (600 mL) , and quenched, washed with saturated NaHCO₃ (150 mL) and H₂O (150 mL) . The organic solution was dried with MgSO₄ and the solvent evaporated prior to silica gel column chromatography. The purified product was collected as a white crystalline solid in yields of 7.4 g (88%) .

¹H NMR (3, 400 MHz, CDCl₃) δ: 6.25 (IH, d, J = 6 Hz, Hl), 4.75

(IH, d, J = 6.4 Hz, H2), 4.29 (IH, ά, J = 6.8 Hz, H3 ) , 4.17

(IH, dd, J = 4.8, 10.4 Hz, H6), 3.95 (IH, t, J = 10.4 Hz IH, H6'), 3.90 (IH, d, J= 10.4 Hz, H4) , 3.84 (IH, dd, J= 4.8, 10.4

Hz, H5), 2.36 (IH, s, OH), 1.07 (9H, s, t-Bu Me), 0.99 (9H, s, t-Bu Me) .

¹³C NMR (3_, 100 MHz, ¹H decoupled 400 MHz, CDCl₃) δ : 144 (Cl), 103 (C2), 77.4 (C4), 72.6 (C5), 70.1(C3), 66.1 (C6), 27.9 (t-Bu Me), 27.4 (t-Bu Me), 23.25 (t-Bu), 20.27 (t-Bu); ESI-MS Calcd. for C₁₄H₂₆O₄Si: 286.4, found: 286.1.

3 -acetyl-1, 5-anhydro-2-deoxy-l,2-C-methylene 4, 6-0- (di-t-butyl- silanediyl) -D-glucal (5). The starting material, 3_, (4.60 g, 16.06 mmol) was dried overnight under vacuum prior to reaction. The starting material was dissolved and stirred to solution with anhydrous Et₂O (56 mL) under a flow of N₂ while on ice (0⁰C) . The reaction was initiated with dropwise addition of 1 M ZnEt₂ in Hex (11 mL, 96.5 mmol) and CH₂I₂ (4 mL, 49 mmol). The reaction was completed after 4 h and this was confirmed by TLC with R_£ : (5:1 v/v Hex:EtOAc) 0.27. The crude reaction mixture was quenched with saturated NH₄Cl (130 mL) and the product extracted with Et₂O, (2 x 200 mL) . The organic solution was washed with saturated NaHCO₃ and brine (2 x 130 mL) and dried over MgSO₄ prior to evaporation and purification of the crude by silica gel column chromatography. The crude product was purified by silica gel flash chromatography with eluent 5:1 Hex: EtOAc and collected as a white solid in yields of 4.42 g (92%). This product, 4,

(4.42 g, 14.71 mmol) was mixed with DMAP (91 mg, 0.74 mmol) and dried overnight under vacuum prior to reaction. At room temperature conditions (22°C) , and with N₂ atmosphere, the reagents were dissolved in pyr (11 mL) and reacted with Ac₂O (3 mL, 30 mmol) for 1 h, until TLC indicated complete conversion to the product with R_f : (5:1 Hex: EtOAc) 0.6. The crude reaction mixture was diluted with Et₂O (220 mL) and quenched, washed with H₂O (2 x 70 mL) . The organic solution was dried with MgSO₄ and evaporated to a viscous oil prior to silica gel column chromatography. The crude product was purified with eluent gradient of 9 to 5:1 Hex: EtOAc, and collected as a viscous oil in yields of 4.6 g (92%).

¹H NMR (5, 400 MHz, CDCl₃) δ: 5.18 (IH, t, J = 4.8 Hz, H3), 4.09 (IH, dd, J = 6.4, 10 Hz, H6), 4.09 (IH, dd, J= 6.4, 10 Hz, H2),

3.69 (IH, t, J= 6.8 Hz, H4 ) , 3.65 (IH, t, J = 6.4 Hz, H6 ' ) , 3.42 (IH, m, H5) , 2.13 (3H, s, OAc), 1.58 (IH, m, Hl), 1.01 (9H, s, t-Bu Me) , 0.98 (9H, s, t-Bu Me) , 0.71 (2H, m, CH₂).

¹³C NMR (5_, 100MHz, ¹H decoupled 400 MHz, CDCl₃) δ : 75.93 (C4) , 75.11 (C3) , 73.5 (C5) , 66.2 (C6) , 55.29 (C2) , 27.95 (t-Bu Me) , 27.51 (t-Bu Me) , 23.23 (t-Bu) , 21.87 (t-Bu) , 16.71 (Cl) , 13.19 (CH₂) ; ESI-MS Calcd for C₁₆H₂₈O₅SiNa : 342.5, found: 343.3. Example 2 : Preparation of Oxepine and Oxepane Nucleosides

General Reaction Procedures and Product Characterization

General procedure for the glycosylation reaction. The starting material (4.7 ramol) was dried overnight under high vacuum. Similarly, in a separate flask, the base (thymine or A^-benzoyl adenine) (24 mmol) and drying reagent (NH₄J₂SO₄ (2.5 mmol) were also dried under vacuum. Under a N₂ atmosphere and at ambient temperatures (22°C) , 85 mL of dry MeCN was added to the flask containing the base and (NH₄) ₂SO₄. Hexamethyldisilazane, HMDS (38 mmols) was added dropwise to the resulting suspension, and the reaction was refluxed for 3 - 4 h until the MeCN-soluble silylated base was formed. The solvent was evaporated and a solution of the starting material in 20 mL of dry MeCN was added and stirred at 22°C under N₂. The reaction was initiated with TMSOTf (1.6 mmol) and completed with reflux (90⁰C) until TLC indicated complete conversion to the produt . The crude reaction mixture was 'worked up' by diluting with EtOAc (150 mL) followed by quenching and washing the upper organic layer with 100 mL each of saturated NaHCO₃ and H₂O . The upper organic layer was dried over MgSO₄ and evaporated to dryness, and the residue was purified on a column of silica gel (eluent 4:1 to 2:1 v/v Hex : EtOAc) .

(IR)-I- [(2,3,4-trideoxy-(5S,6R)-5,7-di-tert-butylsilanediyl)-/?- oxepinyl] thymine (£a) . The product 5.17 was collected as its pure /?-anomer and as a white foam (650 mg, 35%) . R_f : (2:1 Hex: EtOAc) 0.22.

¹H NMR (_6a, 400 MHz, CDCl₃,) δ: 8.56 (bs, NH), 7.19 (IH, d, J = 1 Hz, H6), 5.91 (IH, ddd, J = 2.4, 2.4, 12 Hz, H4 ' ) , 5.74 (IH, dd, J = 6, 10 Hz, Hl'), 5.61 (IH, m, H3 ' ) , 4.59 (IH, dd, J = 2.4, 8.8 Hz, H5'), 4.06 (IH, dd, J = 8.4, 10.5Hz H7 ' ) , 3.85 (IH, dd, J= 8.4, 10.5Hz H7"), 3.64 (IH, m, H6 ' ) , 2.59 (IH, m, H2'), 2.39 (IH, m, H2"), 1.94 (3H, d, J = IHz, H7), 1.05 (9H s, t-Bu Me) , 0.99 (9H s, t-Bu Me) . ¹³C NMR (6a, 100 MHz, ¹H decoupled 400 MHz CDCl₃) δ: 163.4 (CO) , 149.8 (CO) , 140 (C4-) , 135.3, 122 (C3 ' ) , 111.36, 83.81 (Cl¹) , 78.5 (C6') , 77 (C5100¹) , 66.8 (C7¹) , 36.44 (C2 ' ) , 27.82 (t-Bu Me) , 27.40 (t-Bu Me) , 23.02 (t-Bu) , 20.42 (t-Bu) , 13.04; ESI-MS: Calcd for C₂₀H₃₂O₅N₂Si 408.6; found 408.8.

(IR) -1- [ (2,3,4-trideoxy- (5S,6R) -5,7-di-tert-butylsilanediyl)-α- oxepinyl] thymine (6a- a anomer) . The product 6a- a anomer was collected as a mixture of a and /?-anomer and as a white foam (100 mg, 15%) . R_f : (2:1 Hex:EtOAc) 0.20.

¹H NMR (6a- a anomer, 400 MHz, CDCl₃,) δ: 8.56 (bs, NH) , 7.13 (IH, d, J = 1 Hz, H6) , 5.99 (IH, ddd, J = 2.4, 2.4, 12 Hz, H4') , 5.75 (IH, dd, J = 2, 10 Hz, Hl¹) , 5.68 (IH, m, H3 ' ) , 4.67 (IH, dd, J = 2.4, 8.5 Hz, H5 ' ) , 4.24 (IH, dd, J = 8.4, 10 Hz H6') , 4.03 (IH, dd, J = 8.4, 10 Hz H7 ' ) , 3.84 (IH, m, H7 ' ' ) , 2.91 (IH, m, H2 ' ) , 2.33 (IH, m, H2") , 1-88 (3H, d, J = IHz, H7) , 0.98 (9H s, t-Bu Me) , 0.91 (9H s, t-Bu Me) . ESI-MS: Calcd for C₂₀H₃₂O₅N₂Si 408.6; found 408.8.

l(R) -1- [ (2,3,4-trideoxy- (5S,6R) -5,7-di-tert-butylsilanediyl)-β- oxepinyl] -W^-benzoyladenine (^b-)0-anomer) . The product ^b was collected as its pure y0-anomer in yields of 710 mg (30%) and as a white foam R_£ : (2:1 EtOAc : Hex) 0.24.

¹H NMR (6b-/?-anomer, 500 MHz, acetone-d_e) δ: 10.0 (INH, s, H6) , 8.68 (IH, s, H8) , 8.47 (IH, s, H2) , 8-7.2 (5H, m, ar) , 6.02 (IH, d, J= 9 Hz Hl¹) , 5.90 (IH, dd, J = 8, 12 Hz, H3 ' ) , 5.76 (IH, dd, J = 8.5, 11 Hz, H4 ' ) , 4.74 (IH, d, J = 9 Hz, H5 ' ) , 4.04 (IH, ddd, J = 14, 10, 6 Hz, H7 ' ) , 3.91 (IH, dd, J = 9.5, 4.5 Hz, H6') , 3.86 (IH, d, J^" = 18.5 Hz, H7") , 3.40 (IH, d, J = 14 Hz, H2') , 2.84 (IH, m, H2") , 1.07 (9H, s, t-Bu Me) , 1.04 (9H, s, t- Bu Me) .

¹³C NMR (6b-/?-anomer, acetone-d_e, 125.7 MHz, ¹H decoupled 500 MHz) δ: 152.16, 141.65, 139.3 (C3') , 132.6, 131.4, 128.75, 128.5, 128.4, 127.8, 127.6, 123.2 (C4' ) , 84.29 (Cl' ) , 77.84 (C6' ) , 77.31 (C5' ) , 66.58 (CT ) ₁ 35.30 (C2' ) , 27.11 (t-Bu-Me) , 26.78 (t-Bu-Me) , 22.46 (t-Bu) , 19.86 (t-Bu) ; ESI-MS: Calcd. for C₂₇H₃₅N₅O₄SiNa: 544.7, found: 544.1.

1(R)-I- [(2,3,4-trideoxy-(5S,6R) -5,7-di-tert-butylsilanediyl)-α- oxepinyl] -_V*-benzoyladenine (£b- α-anomer) . The product ^b was collected as its pure α-anomer in yields of 375 mg (15%) and as a white foam R_f : (2:1 EtOAc: Hex) 0.18.

¹H NMR (6b- α-anomer , 500 MHz, acetone-d₆) δ : 9.95 (INH, s, H6) , 8.61 (IH, s, H8), 8.51 (IH, s, H2 ) , 8.4-7.4 (5H, m, ar) , 6.31

(IH, d, J = 10.75 Hz Hl') , 5.89 (IH, dd, J = 8, 12 Hz, H3 ' ) ,

5.89 (IH, dd, J = 8.5, 11 Hz, H4 ' ) , 5.14 (IH, d, J = 10.5 Hz,

H6¹), 4.66 (IH, dd, J = 12, 6 Hz, H5 ' ) , 4.04 (IH, d, J = 12 Hz,

H2'), 3.91 (IH, dd, J = 14, 4.5 Hz, H7 ' ) , 3.78 (IH, dd, J = 14.5, 10.5 Hz, H7") , 2.77 (IH, m, H2"), 1.06 (9H, s, t-Bu Me) ,

1.04 (9H, S, t-Bu Me); ESI-MS: Calcd. for C₂₇H₃₅N₅O₄SiNa 544.7; found 544.1.

General procedure for the desilylation reaction.

The starting material, (3.3 mmol) was dried overnight with vacuum. Under N₂ and at 0⁰C, the starting material was dissolved in 7 mL of THF. A solution of 1 M TBAF in dry THF (7 mL) was added with stirring over 5 min. The reaction mixture turned slightly cloudy as the deprotected nucleoside slowly began to precipitate from the solvent. The reaction was complete by TLC after 1 h, so the solvent was removed and the resulting viscous oil was purified by column chromatography (9:1 CH₂Cl₂:MeOH) to give a white foam.

(IR) -1- [(2,3,4-trideoxy-(5S,6R) -5-hydroxy-7-hydroxymethyl) -β- oxepinyl] thymine (£) . The purified product was collected as a white foam in yields of 0.854 g (90%) and with R_f : (9:1 CH₂Cl₂:MeOH) 0.37. ¹H NMR (8, 500 MHz, DMSO-d_e, ) δ : 11.30 (INH, s, H3 ) , 7.57 (IH, s, H6) , 5.74 (IH, t, J = 9Hz, H4 ' ) , 5.60 (IH, d, J = 10Hz, Hl' ) , 5.55 (IH, d, J = 9 Hz, H3 ' ) , 4.05 (IH, d, J = 7.5Hz, H5 ' ) , 3.60 (IH, d, J = HHz H7¹ ) , 3.46 (IH, d, J = HHz, H7") , 3.36 (IH, t, J^" = 6.5 Hz, H6' ) , 3.14 (IH, s, 2'OH) , 2.67 (IH, t , J = 12Hz, H2 ' ) , 2.32 (IH, dd, J = 6, 7.5 Hz, H2") , 1.76 (3H, s, H7) , 1.55 (IH, s, 3'OH) .

¹³C NMR (8, 125 MHz, ¹H decoupled 500 MHz, DMSO-d_ff) δ : 164.22 (CO) , 150.33 (CO) , 138.03 (C3 ' ) , 136.31 (C6) , 83.45 (Cl' ) , 34.89 (C2 ⁽ ) , 122.27 (C4¹ ) , 68.91 (C5¹ ) , 84.80 (C6 ' ) , 61.95 (C7¹ ) , 23.06 (C7) , 108.93 (C5) ; ESI-MS: Calcd. for C₁₂H₁₆N₂O₅: 268.7; found: 269.

(IR) -1- [ (2,3,4-trideoxy- (5S,6R) -5-hydroxy-7-hydroxymethyl)-/?- oxepinyl]

(9) . The purified product was collected as a white foam in yields of 0.184 g (61%) with R_£ : (9:1 CH₂Cl₂ : MeOH) 0.33.

¹H NMR (£, 500 MHz, MeOH-d,) δ : 8.71 (IH, s, H8) , 8.58 (IH, s, H2) , 8.08 (2H, d, ar. ) , 7.65 (IH, m, ar . ) , 7.56 (2H, m, ar) , 6.09 (IH, dd, J = 9.5, 2.5 Hz, Hl' ) , 5.92 (ddd, IH, J = 2.5, 13 Hz, H3 ' ) , 5.76 (IH, m, H4 ' ) , 4.28 (IH, dd, J = 2, 9 Hz, H5 ' ) , 3.89 (IH, dd, J = 4.5, 9.5 Hz, H7 ' ) , 3.79 (IH, ddd, J = 2.5, 5, 9 Hz, H6' ) , 3.68 (IH, dd, J = 6, 11.5 Hz, H7") , 3.17 (IH, m, H2 ' ) , 3.05 (IH, s, 2'OH) , 2.90 (IH, ddd, J = 2, 7, 16.5 Hz, H2") , 1.65 (IH, s, 3'OH) .

¹³C NMR (9, 125.7 MHz, 500 MHz ¹H decoupled, MeOH-d,) δ: 175.23 (CO) , 152.0 (C8) , 142.2 (C2) , 137.6 (C3 ' ) , 132.7 (ar. ) , 128.6 (ar) , 128.2 (ar) , 122.3 (C4 ' ) , 84.90 (Cl' ) , 84.29 (C6- ) , 69.92 (C5' ) , 62.83 (C7' ) , 35.23 (C2 ' ) ; ESI-MS Calcd for C₁₉H₁₉N₅O₄: 402.6; found 404.

General procedure for the hydrogenation reaction.

The product from the desilylation reaction (0.565 mmol) and 152 mg of 10% Pd/C catalyst were dried overnight under vacuum. Dry MeOH (11.5 mL) was added to the evacuated flask and the dark suspension was stirred at room temperature (22°C) . A balloon filled with H₂ was attached to the flask by piercing the septum with a needle. Small aliquots were periodically withdrawn, evaporated to dryness and the extent of the reaction was verified by ¹H NMR. After 4 h, the remaining reaction mixture was filtered and evaporated to dryness. The crude product was purified by flash silica gel column chromatography in 9:1 CH₂Cl₂: MeOH.

(IR) -1- [ (2,3,4-tridβoxy- (5S,6R) -5-hydroxy-7-hydroxymethyl)-/?- oxepanyl] thymine (I₁O-) . The extent of reaction could not be accurately monitored by TLC as the R_£ : 0.21, values for the starting material and product were found to be identical in eluent system 9: 1 CH₂Cl₂: MeOH. The purified product was collected after chromatography in yields of 104 mg (70%) as a white foam.

¹H NMR (10, 500 MHz, MeOH-d₄) δ: 7.56 (IH, s, H6), 5.78 (IH, d, J = 9.5Hz, Hl¹), 3.77 (IH, s, H5 ' ) , 3.70 (IH, d, J= 11.5 Hz, H7'), 3.55 (IH, m, H6'), 3.55 (IH, m, H7"), 1.97 (IH, m, H2 ' ) , 1.90 (2H, m, H3 'H3 " ) , 1.90 (3H, s, H7) , 1.90 (2H, m, 2 'OH, 3 'OH), 1.88 (2H, m, H4'H4"), 1.66 (IH, d, J = 6 Hz, H2").

¹³C NMR (10, 125 MHz, 500 MHz ¹H decoupled, MeOH-dJ δ: 150.9 (CO), 137 (C6), 110 (CO), 95 (C5), 86.64 (C6 ' ) , 86.5 (Cl'), 70.8 (C5⁽), 63.37 (C7'), 34.87 (C4¹), 33.29 (C3 ' ) , 17.9 (C2⁽) 11.18 (C7); ESI-MS Calcd for C₁₂H₁₈N₂O₅Na: 279.3; found 293.1.

(IR) -1- [(2,3,4-tridβoxy- (5S, 6R) -5-hydroxy-7-hydroxymethyl)->S- oxepanyl]

(3Λ) . The extent of reaction could not be accurately monitored by TLC as the R_f : 0.22, values for the starting material and product were found to be identical in eluent system 9:1 CH₂Cl₂ :MeOH. The purified product was collected in yields of 149 mg (99%) as a white foam. ¹H NMR (11, 500 MHz, MeOH- d₄) δ : 9.81 (s, INH) , 8.71 (IH, s, H8) , 8.58 (IH, s, H2) , 8.08 (2H, ar) , 7.65 (IH, ar) , 7.56 (2H, ar) , 6.06 (IH, dd, J = S₁ 5.5 Hz, Hl') , 3.80 (IH, dd, J = 4 , 8,25 Hz, H6') , 3.76 (IH, dd, J^" = 3 , 6 Hz, H4 ' ) , 3.58 (IH, dd, J = 6.35, 12 Hz, H4") , 3.12 (IH, t, J = 8 Hz, H5 ' ) , 2.39 (2H, m, H2 ' H2 " ) , 2.04 (2H, m, H3'H3") , 1.93 (1H, m, H7 ' ) , 1.81 (IH, m, H7") , 1.67 (IH, s, 2'OH) , 1.42 (IH, S, 3'OH) .

¹³C NMR (11, 125.7 MHz, ¹H decoupled 500 MHz, MeOH-dJ δ: 151.9 (C8) , 142 (C2) , 132.7 (ar) , 128.6 (ar) , 128.3 (ar) , 87.25 (Cl') 70.68 (C5¹) , 63.54 (C4⁽) , 52.90 (C6 ' ) , 35.08 (C2¹) , 33.76 (C3') , 18.0 (CT) ; ESI-MS Calcd for C₁₉H₂₁N₅O₄Na: 406.4; found: 406.4.

General Procedure for the tritylation reaction.

The deprotected nucleoside (0.377 mmol) and MMT-Cl (0.44 mmol) were dried overnight under vacuum. Pyridine (1.5 mL) was added at 22°C under nitrogen. The reaction was stirred for 4 h until TLC (9:1 CH₂Cl₂:MeOH) indicated completion. The reaction was diluted with EtOAc (60 mL) and washed with saturated aqueous NaHCO₃ (2 x 60 mL) . The organic layer was then dried over MgSO₄, concentrated and purified by silica gel chromatography with eluent system 20 to 9:1 CH₂Cl₂:MeOH.

(IR) -1- [ (2,3,4-trideoxy- (5S,6R) -5-hydroxy-7- [4-

(methoxyphenyl) diphenyl] ) -/?-oxepanyl] thymine (3J2) . The product was purified and dried as a white foam in yields of 135 mg (66%) and confirmed by TLC R_f : (9:1 CH₂Cl₂: MeOH) 0.68.

¹H NMR (1£, 500 MHz CDCl₃). δ: 8.85 (IH, s, NH), 7.34 (4H, ar) , 7.2 (IH, s, H6), 7.19 (8H, ar) , 6.76 (2H, ar) , 5.74 (IH, dd, J = 3.6, 9.8 Hz Hl'), 3.83 (Hl, ddd, J = 3.6, 4.8, 7.6 Hz, H5'), 3.72 (s, OMe), 3.6 (IH, dd, J= 6, 12.6 Hz, H6 ' ) , 3.31 (IH, dd, J^"= 5.6, 9.6 Hz, H7) , 3.11 (IH, dd, J = 5.6, 9.6 Hz, H7"), 1.98 (IH, dd, J= 3.6, 9 Hz, H3 ' ) , 1.78 (IH, m, H3 " ) , 1.80 (2H, m, H4'H4"), 1.86 (3H, d, J= 1 Hz, H7), 1.62 (2H, dd, J= 8.4, 14.4 Hz , H2 ' H2 " ) ; ¹³C NMR (1£, 125.7MHz, IH decoupled 500MHz, CDCl₃) δ: 163.79 (CO), 158.93 (CO), 149, 144.3,144,1, 135.9 (C6), 135.2, 130.6, 128.5,

128.2, 127.3, 113.5, 110.9, 86.53 (Cl' ) , 87.16 (C5) , 83.58 (C6¹ ) , 73.41 (C5^r ) , 65.90 (C7' ) _# 55.47 (OMe) , 35.81 (C3 ' ) , 33.48 (C4' ) , 18.31 (C2⁽ ) , 12.86 (C7) ; ESI-MS Calcd for C₃₂H₃₄N₂O₆Na: 565.6, found: 565.1.

(IR) -1- [ (2,3,4-tridβoxy- (5S,6R) -5 -hydroxy- 7- [4-

(methoxyphenyl) diphenyl] )-/?-oxepanyl] -.N^-benzoyladenine (^3_) . The product was collected as a white foam in yields of 120 mg (50%) and confirmed by TLC; R_f : (9:1 CH₂Cl₂:MeOH) 0.65.

¹H NMR (]L3, 500 MHz, CDCl₃). δ: 9.03 (IH, s, H4 ) , 8.14 (IH, s, H2), 8.72 (IH, s, H8), 7.95 (2H, ar) , 7.52 (IH, ar) , 7.44 (2H, ar) , 7.29 (3H, ar) , 7.17 (8H ar) , 6.71 (2H, ar.), 5.99 (IH, dd, J = 2.8, 10 Hz, Hl'), 3.90 (IH, t , J = 8.4 Hz, H6 ' ) , 3.79 (IH, dd, J = 5.6, 12.6 Hz, H5 ' ) , 3.71 (OMe), 3.30 (IH, dd, J = 5.6, 9.6 Hz, H4'), 3.14 (IH, dd, J = 6, 9.6 Hz, H4"), 2.27 (IH, ddd, J= 3, 6.5, 15.6 Hz, H2 ' ) , 2.16 (IH, ddd, J= 4.8, 9, 15.6 Hz, H2"), 1.99 (IH, dd, J = 4.8, 17.2 Hz, H3 ' ) , 1.92 (IH, dd, J = 2.8, 17.2 Hz, H3"), 1.82 (IH, ddd, J= 4, 8.4, 16Hz, H7 ' ) , 1.73 (IH, t, J = 8.4 Hz, H7");

¹³C NMR (13, 125.7 MHz, 500 MHz ¹H decoupled, CDCl₃) δ : 158.9 (CO), 152.8 (C8) , 144.3, 140.6 (C2), 144.1, 135.2, 132.98, 130.5, 129.5, 129.1, 128.48, 128.13, 128.1, 128.08, 127.4,

127.3, 113.5, 95, 87.2, 86.44 (Cl'), 83.71 (C5 ' ) , 73.28 (C6 ' ) , 65.88 (C4⁽), 55.44 (OMe), 36.08 (C2'), 33.61 (C7⁽), 18.37 (C3 ' ) ;

ESI-MS Calcd for C₃₉H₃₇N₅O₅: 655.9 found: 655.7.

General procedure for the phosphitylation reaction.

The tritylated nucleoside (0.224 mmol) was dried under vacuum overnight prior to reaction. Dry THF (1.2 mL) was added under N₂. To the resulting solution was added dropwise over a span of 10 min, EtN(I-Pr)₂ (0.89 mmol) and Cl-P (OCEt)N(i-Pr) ₂ (0.246 mmol) . The reaction mixture was stirred for 2 h at 22°C and the reaction progress monitored by TLC (2:1 Hex : EtOAc). The progression of the reaction is also observable by the formation of a white precipitate, C1^~+NH (Et) (iPr) ₂. After the reaction reached completion, EtOAc (15 mL) was added and the mixture was washed twice with saturated aqueous NaHCO₃, dried over MgSO₄ and concentrated to a yellowish foam, which was purified by silica gel chromatography (HexrEtOAc v/v 2:1 to 1:2 with 3% TEA).

(IR) -1- [ (2, 3,4-trideoxy- (5S, 6R) -5-phosphoramidous-7- [4 (methoxyphenyl) diphenyl] ) -yff-oxβpanyl] thymine (14_) . The purified phosphoramidite diastereomers were collected in yields of 131 mg (80%) as a white foam and confirmed by TLC with R_f : (2:1 Hex: EtOAc) 0.52.

³¹P NMR (14, 80.99 MHz, ¹H decoupled 200 MHz, CDCl₃) δ: 149.5 and 149.0; ESI-MS Calcd for C_4IH₅₁N₄O₇PNa: 765.86, found: 765.2 .

(IR) -1- [(2, 3,4-trideoxy- (5S,6R) -5-phosphoramidous-7- [4 (methoxyphenyl) diphenyl] ) -oxepanyl] -W*-benzoyladenine (^5) .

The purified phosphoramidite diastereomers were collected in yields of 135 mg (92%) as a white foam and confirmed by TLC with R_f: (2:1 Hex: EtOAc) 0.33.

³¹P NMR (ljj, 80.99 MHz, ¹H decoupled 200 MHz, CDCl₃) δ: 148.4 and 147.9; ESI-MS Calcd for C₄₈H₅₄N₇O₆PNa 878.9, found 878.3.

Example 3; Synthesis of Modified Oxepane Nucleosides

1- [ (3S,4S) -3,4-epoxy- (5S,6R) -5,7-di-tert-butylsilanediyl) -β- oxepanyl] thymine {16). The starting materials, 6a (200 mg, 0.49 mmol) and mCPBA (88 mg, 0.511mmol) were dried overnight under high vacuum prior to reaction. The reagents were flushed with N₂, and at room temperature (22°C) , anhydrous MeCN (3.5 mL) , CH₂Cl₂ (2.0 mL) were added successively, and the mixture was stirred to solution. The reaction was completed after 24 h and at 40⁰C. The complete conversion of 6a to the product was confirmed by TLC with R_f : (2:1 Hex:EtOAc) 0.19. The reaction mixture was diluted with CH₂Cl₂ (10 mL) and this was quenched, washed with saturated NaHCO₃ (2 x 10 mL) and with H₂O (10 mL) -¹ The organic solution was dried with MgSO₄ and the solvent was evaporated prior to silica gel column chromatography. The crude product, 6.1 was purified with 2:1 to 1:2 Hex: EtOAc which yielded a white crystalline solid, 100 mg (50%) .

¹H NMR (16, 400 MHz, CDCl₃) δ : 9.50 (IH, s, INH), 7.03 (IH, s, H6) , 5.76 (IH, dd, J = 1.6, 11 Hz, Hl'), 4.22 (IH, d, J= 9 Hz, H5'), 4.02 (IH, dd, J = 6.4, 11.2 Hz, H6 ' ) , 3.70 (2H, m, H7',H7"), 3.37 (IH, d, J = 4.8 Hz, H4 ' ) , 3.25 (IH, dd, J= 6.4, 10.6 Hz, H3'), 2.46 (IH, ddd, J= 1.6, 4.8, 15 Hz, H2 ' ) , 2.18 (IH, dd, J= 10.8, 15 Hz, H2"), 1.85 (3H, s, H7) , 0.99 (9H, s, t-Bu Me) , 0.95 (9H, s, t-Bu Me) .

¹³C NMR (16, 125 MHz, ¹H decoupled 500 MHz, CDCl₃) δ : 171.7 (C2), 163.1 (C4) , 134.3 (C6), 110.34 (C5) , 80.85 (Cl'), 74.6 (C5'), 71.9(C7'), 65.1 (C6'), 59.6 (C4'), 50.66 (C3'), 33.38 (C2'), 26.34 (t-Bu Me), 25.99 (t-Bu Me), 22.74 (t-Bu), 11.52 (C7) , 19.13 (t-Bu) ; ESI-MS Calcd for C₂₀H₃₂N₂O₆Si: 424.6, found: 425.3.

1- [ (3S,4S) -3,4-dihydroxy- (5S,6R) -5, 7-di-tert-butylsilanediyl) -β- oxepanyl] thymine (12). The starting materials, 6& (125 mg, 0.3 mmol) , OsO₄ (4 mg, 0.02 mmol) and NMO (150 mg, 1.25mmol) were dried overnight under high vacuum prior to reaction. With N₂ and at room temperature (22°C) , the reagents were dissolved with 5:1 v/v ace: H₂O (5 mL) , and stirred to complete reaction for 5 h. The complete conversion of j>a to the product was confirmed by TLC, with R_£: (2:1 v/v EtOAc:Hex), 0.25. The reaction was quenched with saturated NaHSO₃ (3.8 mL) for an additional 30 min at 22°C. The crude reaction mixture was diluted with CHCl₃ (40 mL) and the organic solution was treated with saturated NaCl (2 x 3OmL) and extracted again with CHCl₃ (3 x 40 mL) . The organic solution was dried with MgSO₄ and evaporated prior to silica gel column chromatography. The crude product was purified with 2:1 EtOAc :Hex and was collected as a white crystalline solid in yields of 65 mg (50%) . ¹H NMR (17, 400 MHz, CDCl₃) δ: 9.34 (IH, s, INH), 7.11 (IH, s, H6) , 6.03 (IH, t, J = 6.4 Hz, Hl'), 4.20 (IH, s, H3 ' ) , 4.06 (IH, m, H5'), 3.96(2H, m, HTHl"), 3.78 (IH, m, H6 ' ) , 3.71 (IH, s, 3'OH), 3.50 (IH, m, H4 ' ) , 2.90 (IH, s, 4'OH), 2.44 (IH, ddd, J = 6.4, 8.8, 15.3 Hz, H2 ' ) , 2.12 (IH, m, H2"), 1.85 (3H s, H7) , 0.99 (9H, s, t-Bu Me) , 0.98 (9H, s, t-Bu Me) .

¹³C NMR (17, 125.7MHz, ¹H decoupled 500 MHz, CDCl₃) δ : 162.8 (C2), 149.0 (C4), 134.93 (C6), 110.3 (C5) , 81.26 (Cl'), 74.29 (C5'), 66.93 (C3'), 65.48 (C6'), 62.90 (C7'), 53.37 (C4'), 36.22 (C2'), 26.05 (t-Bu Me) , 21.62 (t-Bu Me) , 19.04 (t-Bu), 18.92 (t- Bu), 11.55 (C7); ESI-MS Calcd for C₂₀H₃₄N₂O₇SiNa: 465.6, found: 465.2.

1- [ (3S,4S) -3,4-C-methylene- (5S,6R) -5,7-di-tert-butylsilanediyl)- y3-oxβpanyl] thymine {18) . The starting material 6a, (120 mg, 0.294 tnmol) was dried overnight under high vacuum prior to the start of the reaction. Under N₂ and at low temperature (0°C) , 6a, was diluted with anhydrous Et₂O (1.2 mL) and the reaction was initiated with the slow addition (dropwise, 5 min) of 1 M ZnEt₂ in Hex (0.3 rtiL, 2 mmol) and CH₂I₂ (80 μL, 0.9 tnmol). The reaction progress after 24 h and with a gradual temperature increase (0 - 30⁰C) was monitored by TLC which indicated 40% conversion of the starting material to a product with R_f: (5:1 v/v Hex : EtOAc) 0.14. The crude product was extracted in Et₂O (5 mL) , treated with saturated NH₄Cl (2.5 mL) and extracted, washed again with Et₂O (4 mL) and H₂O (2.5 mL) , saturated NaCl (2.5 mL) . The organic solution was dried with MgSO₄ and the solvent was evaporated prior to silica gel column chromatography. The crude product was purified with 5:1 to 2:1 Hex:EtOAc, as a white crystalline solid in yields of 35 mg (30%) .

¹H NMR (lji, 400 MHz, CDCl₃) δ : 7.12 (IH, s, H6) , 5.85 (IH, d, J = 12 Hz, H4') , 5.73 (IH, dd, J = 1.6, 10 Hz, Hl') , 5.62 (IH, m, H3' ) , 4.53 (IH, dd, J = 2.4, 9.4 Hz, H5 ' ) , 3.99 (IH, dd, J = 4.8, 10.4 Hz, H7') , 3.79 (IH, dd, J = 10.4,14.8 Hz, H7") , 3.57 (IH ,dd, J = 4.8, 9.4 Hz, H6 ' ) , 3.28 (2H, s, CH₂) , 2.52 (IH, ddd, J = 2.8, 13.4, 14.6 Hz, H2 ' ) , 2.31 (IH, ddd, J = 8.8, 14.6 Hz, H2") , 1-88 (IH, d, H7) , 0.98 (IH, s, t-Bu Me) , 0.92 (IH, s, t-Bu Me) .

¹³C NMR (18, 125.7MHz, 500MHz ¹H decoupled, CDCl₃) δ: 162.4 (C2), 149.6 (C4), 138.8 (C4'), 132.0 (C6), 120.97 (C3'), 109.2 (C5), 83.31 (Cl'), 77.14 (C6'), 76.26(C5'), 65.5 (C7^#), 35 (C2'), 26.99 (>C), 26.38 (t-Bu Me), 25.97 (t-Bu Me), 21.52 (t-Bu), 18.96 (t-Bu), 12.31 (C7); EI-MS Calcd for C₂₁H₃₄N₂O₅Si: 422.6, found: 422.

1- [2,3-didβoxy- (4R) -4-hydroxy- (5S,6R) -5,7-di-tert- butylsilanediyl)-/?-oxepanyl] thymine (3J)) . The starting material, ^a, (25 mg, 0.06 mmol) was dried overnight under high vacuum prior to initiating the reaction the following day. With N₂ and at low temperature (0⁰C) , the starting material was dissolved with anhydrous THF (0.3 mL) and the reaction was initiated with the dropwise addition of BH₃-THF (20 μL, 0.21 mmol) and stirred to completion for 20 h with a gradual temperature increase (0 - 22°C) . The extent of the reaction was monitored by TLC with eluent system 2:1 v/v EtOAc :Hex which indicated a product with R_f: 0.30. The reaction was placed in an ice bath and treated with H₂O (0.5 mL) , 3 M NaOH (0.15 mL) and 30% H₂O₂ (0.15 mL) . This mixture was stirred for and additional 2 h, or until TLC indicated complete reaction, R_£: (2:1 EtOAc: Hex) 0.36. The reaction mixture was diluted with Et₂O (3 mL) , treated with saturated NaCl (2 x 0.15 mL) and the crude product was extracted in Et₂O (3 x 3 mL) . The organic solution was dried with MgSO₄ and evaporated prior to silica gel column chromatography. The crude product was purified by chromatography with eluent system, 2:1 EtOAc : Hex, and collected as a white crystalline solid in yields of 17 mg (67%) .

¹H NMR (19, 500 MHz, CDCl₃) δ : 8.16 (IH, s, IH), 7.09 (IH, s, H6) , 5.80 (IH, t, J= 6.5 Hz, Hl'), 4.05 (IH, dd, J= 5.5, 10.8 Hz, H7' ) , 3.77 (IH, t, J = 10.5 Hz, H7" ) , 3.61 (IH ,m, J = 5 Hz, H5' ) , 3.56 (IH, dt, J = 1.5, 10 Hz, H4 ' ) , 3.46 (IH, dt , J = 6.5, 8.8 Hz, H6' ) , 2.95 (IH, S, 4'OH) , 2.10 (IH, m, H2 ' ) 1.94 (IH, m, H2") , 1.72 (2H, m, H3'H3") , 1.87 (IH, s, H7) 1 (9H, s, t-Bu Me) , 0.93 (9H, s, t-Bu Me) .

¹³C NMR {19_, 125.7MHz, 500MHz ¹H decoupled, CDCl₃) δ : 153.13 (C4) , 134.80 (C6) , 110.2 (C5) , 82.19 (Cl' ) , 81.26 (C4' ) , 74.20 (C5' ) , 65.45 (C6' ) , 65.45(C7' ) , 28.43 (C2' ) , 26.42 (t-Bu Me) , 25.95 (t-Bu Me) , 24.93 (C3^; ) , 21.63 (t-Bu) , 18.93 (t-Bu) , 11.59 (C7) ; EI-MS Calcd for C₂₀H₃₄N₂O₆Si : 426.6, found : 427.3.

Example 4; Oligonucleotide Synthesis and Characterization

Reagents. The reactions for the preparation of the nucleoside monomers and their automated assembly into oligonucleotides are sensitive to moisture and require anhydrous glassware, reagents and handling. The solvents and reagents for phosphoramidite preparation, nucleoside derivitization to the solid support and oligonucleotide syntheses were of the highest quality and kept completely anhydrous during use.

Derivatization of the Solid Support. The following procedure was used to couple the tritylated oxapane nucleoside monomers with the CPG support. Into a dried 5 mL glass vial was added the tritylated nucleoside monomer (0.7 mmol) with coupling reagents O- (7-azabenzotriazol-l-yl) -N₁N₁N' ,N' -tetramethyluronium hexafluorophosphate (HATU), or 0- (Benzotriazol-1-yl) -N₁N,N' ,JV'- tetramethyluronium hexafluorophosphate (HBTU) (0.1 mmol), and -V,iV-dimethylaminopyridine (DMAP) (0.1 mmol) and the solid support [succinyl linked 500 A long chain alkyl amino controlled pore glass i.e. succinyl linked LCAA CPG (250 mg) ] prepared according to literature procedure [Pon, R. T. et . al . (2001), Rapid Esterification of Nucleotides to Solid Supports for Oligonucleotide Synthesis Using Uronium and Phosphonium Reagents, Bioconj . Chem. 10, 1051-1057.]. The reaction was completed at room temperature (22°C) with N₂ atmosphere in anhydrous MeCN (1 mL) . The extent of the coupling reaction is contingent with the reaction time (20-40 min produces low nucleoside loading 20-40 μmol/g while longer reaction times can yield higher loadings of ca. 80 μmol/g) . The nucleoside derivatized solid support was filtered, washed successively with 25 mL of DCM, 50 mL of MeOH and additional 25 mL of DCM prior to determining the nucleoside loading. The nucleoside loadings were determined by spectrophotometric mono- and dimethoxytrityl cation colorimetric assay. The support was dried in-vacuo for 24 h before use, loaded into an empty synthesizer column with replaceable filters (ABI) , crimped closed with aluminum seals (ABI), and installed on the instrument. Alternatively, the solid support described by Manoharan and co-workers [Unylinker™ support LCAA CPG; available through ChemGenes, Inc.] was used for the automated synthesis of DNA, RNA and oxepane modified oligonucleotides [Manoharan, M. et. al . (2003), J. Am. Chem. Soc . , 125, 12380-12381.] . This is a very useful solid support because the tritylated nucleoside phosphoramidites can be coupled directly (without prior derivatization of the support with the first nucleoside unit) to the Unylinker™ support.

Automated Solid Phase Synthesis of Oligonucleotides. The solid phase syntheses of oligonucleotides were conducted on either an ABI 3400 or 381A gene machine synthesizer. The reagents for the solid phase synthesis procedure included: 1) the detritylation reagent (3% solution of TCA in DCM) , 2) the coupling reagent (0.25 M ethylthiotetrazole in acetonitrile or 0.25 M ETT in MeCN), 3) the capping reagents (Cap A : 1:1:8 v/v/v Ac₂O :pyr: THF, and Cap B: 10% N-methyl imidazole in THF or AT-Me Im in THF), 4) the oxidation reagents (0.1 M iodine in 75:20:5 v/v/v THF :pyr: H₂O) and 5) acetonitrile wash (Biotech grade purchased from EMI with low water content and 99.999% purity). These reagents were purchased and used as anhydrous reagents and solvents from Chemgenes Inc. The oligonucleotide syntheses were performed on 0.3 - 1 μmol scales with 500 A succinyl linked LCAA CPG derivitized with the tritylated nucleoside monomers or directly on the universal linker LCAA. The modified nucleoside phosphoramidites were prepared in 0.05-0.15 M solutions with anhydrous CH₂Cl₂, or MeCN. The coupling times for these were extended to 30 min with 0.25 M ETT in MeCN as activator and the detritylation time was also extended to 2.5 min. For the conventional DNA and RNA syntheses, DNA phosphoramidites were prepared as 0.1 M solutions in anhydrous MeCN and coupled to the solid support for 2 min. The RNA amidites were prepared as 0.15 M solutions in anhydrous MeCN and the coupling times were extended to 10 min reactions (rG amidites required 15 min coupling times) .

The assembly of oligonucleotides was performed with the following sequential synthesis steps: 1) detrityaltion-. a DCM wash step for 40 s followed by 3% TCA in DCM delivery for 120 s which cleaves the trityl protecting group that can be used for determining coupling yields by UV spectroscopic quantitation (DMT⁺: λ: 504 nm, e: 76 000 L/mol cm^"1 and MMT⁺: λ: 478 nm, e: 56 000 L/mol cm^"1), 2) coupling: a delivery of p'hosphoramidite dissolved in CH₂Cl₂ or MeCN (0.05 - 0.15 M solutions) with the activator (0.25 M ETT in MeCN) for a coupling time of 90 s (DNA) , 10 or 15 min (RNA) and 30 min (modified nucleoside amidites) , 3) capping: the delivery of Cap A and Cap B for 15 s and a wait time for 45 s followed by, 4) oxidation: the delivery of the oxidant (0.1 M iodine in 75 : 20 : 5 v/v/v THF : pyr : water) for 20 s and an additional wait step for 20 s. Prior to the oligonucleotide assembly, the derivitzed support was capped [capping cycle provided by manufacture) to block the undesired reactive sites.

Complete Deprotection and Purification of Oligonucleotides . The

CPG bound oligonucleotide sequences were dried with argon in the synthesizer column (10 min) and transferred into an autoclaved,

1.5 mL screw cap microtube. The oligomer bound support, was treated with a 1 mL solution of 3:1 v/v ammonium hydroxide

(NH₄OH) in absolute EtOH. The cleavage reaction of the oligomer from the support and the protecting group deprotection reactions were performed at 55°C for 4 - 6 h, and for mixed base sequences for 16 - 24 h to ensure the complete removal of the oligonucleotide base protecting groups. After cleavage and deprotection, this solution was evaporated to dryness and the oligomer was re-suspended in autoclaved water for determining the yield by UV absorbance measurements.

Alternatively, oligoribonucleotides (RNA) and oxepane modified RNAs require a 2 ' -desilylation reaction. This was performed with 300 - 500 μL of anhydrous triethylamine trihydrofluride, TREAT HF, (Aldrich) on an over-head shaker for 48 h at ambient temperature (22°C) . Alternatively, a faster desilylation procedure was followed by adding 0.3 mL of a solution of 0.75 mL NMP, 1.0 mL TEA and 1.5 mL TREAT HF at 65 ⁰C for 90 min. After complete reaction, the oligoribonucleotide was precipitated directly with 25 μL of a 3 M NaOAc solution and 1 mL n-BuOH. The precipitation process was optimized with dry ice for 2 h, centrifuged and the supernatant removed prior to dissolving the crude oligomer in autoclaved water. The recovery of crude oligoribonucleotide sequence from precipitation was determined by UV absorbance and this was followed by purification of the crude. Oligonucleotides were analyzed and purified by 24% denaturing polyacrylamide gel electrophoresis or Anion Exchange HPLC using a 30% gradient of IM LiClO₄. The purified samples (from PAGE and AE HPLC) were desalted from water soluble counterions and lower molecular weight impurities by gel filtration with Nap^® 10 or 25 size exclusion chromatography columns containing Sephadex^* G-25 Superfine medium (Amersham Inc.), prepared by cross-linking dextran with epichlorohydrin. This formed a gel in autoclaved water and was used to elute the purified oligonucleotide samples (ca. 0.5 - 1 A₂₆₀ units in 1 mL autoclaved water with Nap" 10 columns and ca. 15 - 20 A₂₆₀ units in 2.5 mL autoclaved water with Nap" 25 columns) in 1 mL fractions collected in sterile 1.5 mL microtubes. Example 5 ; Monitoring Duplex Formation

via Ultraviolet Spectroscopy.

UV Thermal Denaturation Studies. Oligonucleotide hybridization experiments were performed on a Varian Cary I or 300 UV-VIS spectrophotometer equipped with a Pelltier temperature controller interfaced to a PC running Windows^* based software (Win 3.1 or Win 2000 Professional). The thermal melt experiments for hybridized complementary oligonucleotide strands were monitored by hyperchromic changes in the UV absorbance at 260 nm (and 284 nra for triplex studies) with increasing temperatures (5 - 80⁰C) . The melting curves were collected with a data interval and temperature gradient of 0.5°C/min under constant N₂ flow to prevent condensation at lower temperature. Molar extinction coefficients (e_2So) for the single strands were calculated based on those of the mono- and di-nucleotides using the nearest neighbor approximation method of Puglisi and Tinoco [Puglisi, J. D. et . al . (1989) Methods Enzymol . 180, 304-325] . The melting temperature, (TJ for the complexes was calculated from the first derivative plots of the melting curve, which produces a maximum value corresponding to the inflection point of the melting transition and represents the temperature at which 50% of the complex has disassociated. The spectra were acquired in duplicate scans to ensure reproducibility. The absorbance versus temperature data were converted to ASCII binary format and imported into a spreadsheet with the Microsoft Excel™ XP Software for further manipulation. Hyperchromicity values (% H) were calculated as relative changes in the absorbance (260 nm) at a given temperature. This was calculated with the equation: % H = [ (A_τ - A₀) /A_f] x 100, where H is the hyperchromicity, A_τ is the absorbance at a given temperature (T) , A₀ is the initial absorbance at the start temperature and A_£ is the absorbance at the final temperature.

Samples for T_m studies were generally prepared by evaporating an equimolar mixture of complementary strands to dryness with a Speed" Vac concentrator and then re-dissolving them in 1 mL of the appropriate buffer for a duplex concentration of 3 - 5 μM. The buffer typically consisted of a physiologically relevant phosphate buffer (i.e. 140 τnM KCl, 1 mM MgCl₂, 5 mM Na₂HPO₄ adjusted to pH: 7.2). The component singles strands (1.5 - 2.5 μM) were also analyzed by T_n, experiments. The solutions were heated at 90⁰C for 10 - 15 min to denature the complex, and then slowly cooled to room temperature for 1.5 - 2 h and annealed overnight (12 - 16 h) at 4°C prior analysis. The hybridized samples were quickly transferred into pre-chilled (on ice) Hellma^* QS- 1.000 quartz cells and sealed with stopper and conserved with parafilm to prevent solvent evaporation during the thermal analysis. The solutions were degassed by sonication for 5 - 10 sec and further equilibrated at 5°C for 5 min in the cell chamber with N₂ flow prior to the analysis. The N₂ was continuously flushed through the chamber to prevent condensation at low temperatures (5 - 25°C) .

UV Stoichiometric Studies (mixing curves or Job plots) . The proportion in which (complementary) strands associate can be determined by monitoring the relative change in absorbance values at a given wavelength (260 nm) with titration of a solution containing one strand to an equimolar solution of the second complementary strand [Pilch, D. S. et . al . (1990) Proc. Natl. Acad. Sci . USA, 87, 1942-1946.]. This study was conducted on a UV-VIS Cary 300 dual beam spectrophotometer. Equimolar stock solutions of each oligonucleotide strand (2.5 nmol) were prepared in 0.5 mL of buffer (10 mM Na₂HPO₄, 100 mM NaCl, pH: 7.2 or 10 mM Na₂HPO₄, 50 mM MgCl₂ pH: 7.3). At low temperature (5°C) , 100 μL aliquots (0.5 nmol) of T/U solution was titrated into the stock solution containing A, and allowed to hybridize for 5-10 min prior to measuring the UV absorbance at 260 nm under constant flow of N₂. Absorbance values were measured at 260 nm for each mol fraction of T titrated into a fixed concentration of a complementary A stock solution to determine the stoichiometry of the hybridization interaction. Both the native and oxapane-modified hybrids show duplex formation under these conditions (1:1 stoichiometry) (Figure 2). Example 6; Monitoring Duplex Formation via Circular Dichroism

Spectroscopy

Samples for the CD experiments were prepared similarly to the T_a experiments with typical phosphate buffers (i.e. 3 - 5 μM complex in 140 mM KCl, 1 mM MgCl₂, 5 mM Na₂HPO₄ adjusted to pH: 7.2). The component singles strands (1.5 - 2.5 μM) were also analyzed by the CD experiments. Since CD is also useful in monitoring the melting of hybrid complexes, the relative change in ellipticity (mdeg) at a given wavelength (310 - 200 nm) with temperature (5 - 80⁰C) produced a thermal transition curve from which T_m values were determined. Equimolar mixtures of the single strands were annealed by pre-heating the solution (90°C) for ca. 10 min and cooling to room temperature (22°C) for approximately 2 h and at 4°C for 12 - 16 h. The samples were transferred to Hellma QS-I.000 fused quartz cells and maintained within the sample cell holder at 5°C with N₂ for 10 - 15 min prior to the spectral acquisition. The CD spectra were collected on a Jasco J-710 spectropolarimeter equipped with a thermoelectrically controlled external constant temperature (NESLAB^* RTE-111 circulating bath) . Samples were dispensed in cells (Hellma⁰" QS- 1.000 quartz cells) with a 1 cm path length. Before the CD spectra were determined, the samples were equilibrated at a given temperature for approximately 5 min with N₂. Each spectrum was collected as an average of 3 scans at a rate of 100 nm/τnin and band width of a 1 nm interval. The sampling wavelength was adjusted to 0.2 nm and the spectra were analyzed between 350 and 200 nm. The raw data was processed using J-700 Windows^* software (version 1.00) as supplied by the manufacture, and was normalized by subtraction of the buffer, noise reduction (i.e. line smoothing), and concentration such that the molar ellipticity was calculated from the equation [0] = θ /cl, where β is the relative ellipticity (mdeg) , c is the molar concentration of the oligonucleotides (M) and 1 is the path length of the cell (cm) . The data was imported and processed with Microsoft Excel™ XP spreadsheet after manipulation with J-700 Windows* software (version 1.00).

Example 7; Nuclease Resistance of Oxepane Oligonucleotides

To the oligothymidylate (0.7 ODs) was added 300 μL of 10% FBS (0.2 μM, in 10% fetal bovine serum diluted with eagle's medium) and incubated at 37°C up to 24 h incubation. For each time point, a 50 μL aliquot of the reaction mixture was removed and frozen on dry ice for 10 min to stop the reaction, followed by evaporation in a Speed-Vac concentrator. Aliquots were re- dissolved in deionized formamide and analyzed by denaturing 24% PAGE, and visualized by Stains-All" dye. The data show that the oxepane thymidine oligonucleotide is exceedingly resistant towards nuclease degradation (Figure 5) .

Example 8 ; RNase H Assays

An aliquot (7 pmol) of the target radiolabeled 5'-³²P-RNA strand (200 pmol) was added with a complementary (1.8 fold excess) antisense single strand and annealed with 5x reaction buffer (20 pmol in 10 μL buffer; 100 mM Tris HCl pH: 7.5, 100 mM KCl, 50 mM MgCl₂, 0.5 mM EDTA, 0.5 mM DTT) and autoclaved water (100 μL, total volume) . The complementary sequences were denatured for 5 - 10 min at 95°C, slowly cooled to room temperature for 1.5 - 2 h and annealed overnight for 12 - 16 h at 4°C prior to the enzymatic reaction. E. coli RNaseH assays were performed at 20⁰C in 10 μL reactions containing 2 pmol of duplex substrate with 2 μL of reaction buffer (5x RNaseH buffer, Amersham Biosciences Inc.) and 0.5 μL of E. coli RNaseH enzyme (Amersham Biosciences Inc., concentration of 5 units/μL in storage buffer 20 mM Tris HCl pH: 7.9, 100 mM KCl, 10 mM MgCl₂, 0.1 mM EDTA, 0.1 mM DTT and 50% glycerol) . Each reaction was quenched at various time points by heating to 90⁰C and with the addition of "stop solution" (10 μL, 50 mM EDTA in formamide with BPB and XC dyes) prior to analysis by 16% PAGE that was run for 2.5 h at 2000 V, 30 mA and 55 W. The reactions were analyzed and visualized by autoradiography. The extent of the cleavage reaction for the radiolabeled RNA portion of the RNA/antisense hybrid was determined quantitatively by densiometric analysis (UN-SCAN- IT™ software) with the disappearance of the full length RNA and/or the appearance of the smaller RNA degradation products (Figure 6) .

Example 9: Gene Silencing through RNA Interference (RNAi)

HelaXl/5 cells that stably express firefly luciferase were grown as previously described [Watts et al . Nucleic Acids Res. (2007), 35, 1441-1451] . The day prior to transfection, 0.5 x 10⁵ cells were plated in each well of a 24 -well plate. The next day, the cells were incubated with increasing amounts of siRNAs premixed with lipofectamine-plus reagent (Invitrogen) using 1 μL of lipofectamine and 4 μL of the plus reagent per 20 pmol of siRNA (for the highest concentration tested) . For the siRNA titrations, each siRNA was diluted into dilution buffer (30 mM HEPES-KOH, pH 7.4 , 100 mM KOAc, 2 mM MgOAc₂) and the amount of lipofectamine-plus reagent used relative to the siRNAs remained constant 24 hours after transfection, the cells were lysed in hypotonic lysis buffer (15 mM K₃PO₄, 1 mM EDTA, 1% Triton, 2 mM NaF, 1 mg/ml BSA, 1 mM DTT, 100 mM NaCl, 4 μg/mL aprotinin, 2 μg/mL leupeptin and 2 μg/mL pepstatin) and the firefly light units were determined using a Fluostar Optima 96 -well plate bioluminescence reader (BMG Labtech) using firefly substrate as described [Novae, 0. et al . Nucleic Acids Res. (2004) 32, 902- 915] . The luciferase counts were normalized to the protein concentration of the cell lysate as determined by the DC protein assay (BioRad) (Figure 7) .

Although preferred embodiments have been described herein, a person skilled in the art would understand the variations that may be made thereto without departing from the spirit of the invention or the scope of the appended claims. All reference disclosed herein are hereby incorporated by reference.

Claims

Claims :

1. An oxepane nucleoside of formula (I) :

wherein,

B is a heterocyclic base,-

R₀ is selected from the group consisting of hydrogen and alkyl having 1 to 8 carbons; and

R₁ through R₁₀ are independently selected from the group consisting of a hydrogen, azido, amino, allyl, carboxyl, ester, halogen (fluorine, chlorine, bromine, and iodine) , hydroxyl, nitrile, sulfhydryl, alkyl, hydroxylmethyl , alkylhalide, alkoxy, alkoxyalkyl, alkylsulfhydryl, allyl, propargyl, ethynyl, and ethenyl ;

wherein any two of R₀ through R₁₀ may be covalently bonded to adjacent or non-adjacent carbons l'-6' to form a bicyclic structure; the covalently bonded group selected from the group consisting of -0-, -CH₂-, -CH₂CH₂-, -CH₂CH₂CH₂-, -OCH₂-, -OCH₂CH₂- , -CH₂OCH₂-,-NH-, -NHCH₂-, -NHCH₂CH₂-, -CH₂NHCH₂-, -S-, -SCH₂CH₂-, and -SCH₂CH₂CH₂- ; and

mirror image enantiomers thereof.

2. The oxepane nucleoside of claim 1, wherein B is capable of base pairing.

3. The oxepane nucleoside of claim 2, wherein B is selected from the group consisting of thymine, uracil, cytosine, adenine, guanine, inosine, 5-methylcytosine, 2-thiothymine, 4- thiothymine, 7-deazaadenine, 9-deazaadenine, 3-deazaadenine, 7- deazaguanine, 9-deazaguanine, 6-thioguanine, isoguanine, 2,6- diaminopurine, hypoxanthine , and 6-thiohypoxanthine;

4. The oxepane nucleoside of claim 1, wherein B is selected from the group consisting of 5-fluorocytosine, 5-bromocytosine,

5-iodocytosine, isocytosine, l^-methylcytosine, 5-iodouracil, 5- fluorouracil, 4-thiouracil, 2-thiouracil, (E) -5- (2- bromovinyl) uracil, N⁶-methyladenine, 2-chloroadenine, 2- fluoroadenine, 2-chloroadenine, N6-cyclopropyl-2, 6- diaminopurine, nicotinamide, 2-aminopurine, 1, 2 ,4-triazole-3- carboxamide .

5. The oxepane nucleoside of any one of claims 1-3, wherein the alkyl group is selected from the group consisting of methyl, ethyl, propyl, butyl, and functionalized alkyl groups thereof.

6. The oxepane nucleoside of claim 5, wherein the functionalized alkyl group is selected from the group consisting of methylamino, dimethylamino, ethylamino, diethylamino, propylamino and butylamino groups.

7. The oxepane nucleoside of any one of claims 1-3, wherein the alkoxy group is selected from the group consisting of methoxy, ethoxy, propoxy and functionalized alkoxy groups thereof .

8. The oxepane nucleoside of claim 7, wherein the functionalized alkoxy group is selected from the group consisting of -O(CH₂)_q-R, where q=2-4 and R is -NH₂, -OCH₃, or - OCH₂CH₃.

9. The oxepane nucleoside of any one of claims 1-3, wherein the alkoxyalkyl group is selected from the group consisting of methoxyethyl , and ethoxyethyl .

10. The oxepane nucleoside of any one of claims 1-3, wherein R₀ is hydrogen.

11. The oxepane nucleoside of claim 1, having the structure of formula (II) :

wherein,

B is selected from the group consisting of thymine, uracil, cytosine, adenine, guanine, inosine, 5-methylcytosine, 5- fluorocytosine, 5-bromocytosine, 5-iodocytosine, isocytosine, N⁴-methylcytosine, 5-iodouracil, 5-fluorouracil, 4-thiouracil, 2-thiouracil, (E) -5- (2-bromovinyl) uracil, 2-thiothymine, 4- thiothymine, 7-deazaadenine, 9-deazaadenine, N⁶-methyladenine, 2-chloroadenine, 2-fluoroadenine, 2-chloroadenine, isoguanine, 3-deazaadenine, 7-deazaguanine, 9-deazaguanine, 6-thioguanine, nicotinamide, 2-aminopurine, 2 , 6-diaminopurine, N6-cyclopropyl- 2 , 6-diaminoρurine, hypoxanthine, 6-thiohypoxanthine, 1,2,4- triazole-3-carboxamide .

12. The mirror image enantiomer of the oxepane nucleoside of claim 11.

13. An oxepane nucleotide comprising the oxepane nucleoside of any one of claims 1-3 and 5-12.

14. An oxepane nucleotide comprising the oxepane nucleoside of claim 4.

15. An oligonucleotide comprising a plurality of nucleotides covalently bonded through a phosphorus containing moiety, wherein at least one nucleotide is the oxepane nucleotide of claim 13.

16. The oligonucleotide of claim 15, wherein the phosphorus containing moiety is selected from the group consisting of phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, and phosphoramidate internucleoside linkages.

17. The oligonucleotide of claim 16, wherein the oligonucleotide is 15-50 nucleotides in length.

18. The oligonucleotide of claim 17, wherein the oligonucleotide is capable of hybridizing to and inducing RNase H-mediated cleavage of an RNA strand.

19. A double stranded oligonucleotide comprising at least one oligonucleotide of claim 15.

20. The double stranded oligonucleotide of claim 19, where one or both strands have overhangs from 1-5 nucleotides on the 3'- end.

21. The double stranded oligonucleotide of claim 19, wherein neither strand has an overhang.

22. The oligonucleotide of claim 15, wherein the oligonucleotide is 15-80 nucleotides in length and exhibits self-complementarity thereby being capable of adopting a hairpin duplex structure.

23. A method for increasing at least one of therapeutic efficacy, nuclease stability, and/or selectivity of binding of an oligonucleotide, the method comprising inserting or replacing at least one nucleotide of the oligonucleotide with an oxepane nucleotide.

24. A method of selectively modulating gene expression by administering the oligonucleotide of any one of claims 15-18 and 22.

25. A method of selectively modulating gene expression by administering the double stranded oligonucleotide of any one of claims 19-21.

26. A pharmaceutical composition comprising the oligonucleotide according to any one of claims 15-18 and 22 and a pharmaceutically acceptable carrier.

27. A pharmaceutical composition comprising the double stranded oligonucleotide according to any one of claims 19-21 and a pharmaceutically acceptable carrier.

28. A pharmaceutical composition comprising the nucleosides of any one of claims 1-12 and a pharmaceutically acceptable carrier.

29. A pharmaceutical composition comprising the nucleotide according to any one of claims 13-14 and a pharmaceutically acceptable carrier.

30. Use of the oligonucleotide according to any one of claims 15-18 and 22 for modulating gene expression.

31. Use of the double stranded oligonucleotide according to any one of claims 19-21 for modulating gene expression.

32. Use of the oligonucleotide according to any one of claims 15-18 and 22 in the preparation of a medicament for modulating gene expression.

33. Use of the double stranded oligonucleotide according to any one of claims 19-21 in the preparation of a medicament for modulating gene expression.

34. A compound selected from the group consisting of compounds of formulas (III) , (IV) , (V) and (VI) :

III

wherein B is selected from the group consisting of thymine, uracil, cytosine, adenine, guanine, inosine, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, isocytosine, N⁴-methylcytosine, 5-iodouracil, 5-fluorouracil, 4-thiouracil, 2-thiouracil, (E) -5- (2-bromovinyl) uracil, 2-thiothymine, 4- thiothymine, 7-deazaadenine, 9-deazaadenine, N⁶-methyladenine, 2-chloroadenine, 2-fluoroadenine, 2-chloroadenine, isoguanine, 3-deazaadenine, 7-deazaguanine, 9-deazaguanine, 6-thioguanine, nicotinamide, 2-aminopurine, 2, 6-diaminopurine, N6-cyclopropyl- 2 , 6-diaminopurine, hypoxanthine, 6-thiohypoxanthine, and 1,2,4- triazole-3-carboxamide,

and mirror image enantiomers thereof.

35. A method of preparing an oxepane nucleoside comprising the step of reducing the double bond of an oxepine nucleoside with a reducing agent.

36. The method of claim 35 wherein the double bond is between the 3' carbon and the 4' carbon.

37. The method of claim 36 wherein the reducing occurs in the presence of a palladium metal pre-catalyst .

38. The method of claim 37 wherein the palladium metal pre- catalyst is palladium/charcoal.

39. The method of claim 35, the oxepine nucleoside is produced by deprotecting a protected oxepine nucleoside.

40. The method of claim 39, wherein the protected oxepine nucleoside is protected at the functional groups attached to the 5' carbon and the 6' carbon.

41. The method of claim 40, wherein the protected oxepine nucleoside is protected by a silyl group.

42. The method of claim 39, wherein the protected oxepine nucleoside is produced by glycolsyating a nucleobase with a protected cyclopropanated hexopyranose .

43. The method of claim 42, wherein the nucleobase is protected.

44. The method of claim 43, wherein the nucleobase is protected with a silyl group.

45. The method of claim 42, wherein glycosylation occurs via a Vorbruggen-like glycosylation reaction.

46. The method of claim 42, wherein the cyclopropanated hexopyranose is protected via silylation.

47. The method of claim 46, wherein the cyclopropanated hexopyranose is acetylated.

48. The method of claim 42 wherein the protected cyclopropanated hexopyranose is comprised of a sugar selected from the group consisting of a D-glucal and L-glucal .

49. The method of claim 42 wherein the cyclopropanated hexopyranose is acetylated 5' , 6' -O-bis-siloxane-protected D- glucal .

50. The method of claim 47, wherein the cyclopropanated hexopyranose is acetylated at the 3' position.

51. The method of claim 49, wherein the protected cyclopropanated hexopyranose is produced by cyclopropanating a protected unsaturated hexopyranose sugar .

52. The method of claim 50, wherein the cyclopropanation of the protected sugar occurs under Simmons-Smith conditions.

53. The method of claim 42, wherein the nucleobase is selected from the group consisting of thymine, uracil, cytosine, adenine, guanine, inosine, 5-methylcytosine, 5-fluorocytosine, 5- bromocytosine, 5-iodocytosine, isocytosine, N⁴-methylcytosine, 5-iodouracil , 5-fluorouracil, 4-thiouracil, 2-thiouracil, (E) -5- (2-bromovinyl) uracil , 2-thiothymine, 4-thiothymine, 7- deazaadenine, 9-deazaadenine, N^s-methyladenine, 2-chloroadenine, 2-fluoroadenine, 2-chloroadenine, isoguanine, 3-deazaadenine, 7- deazaguanine, 9-deazaguanine, 6-thioguanine, nicotinamide, 2- aminopurine, 2 , 6-diaminoρurine, N6-cyclopropyl-2, 6- diaminopurine, hypoxanthine , 6-thiohypoxanthine, and 1,2,4- triazole-3-carboxamide .

54. The method of claim 47 wherein acetylation of the cyclopropanated hexopyranose proceeds as:

55. The method of claim 54 wherein acetylation of the cyclopropanated hexopyranose proceeds as :

56. The method of claim 51 wherein cyclopropanation of the protected, unsaturated hexopyranose proceeds as :

57. The method of claim 56 wherein cyclopropanation of the protected, unsaturated hexopyranose proceeds as:

58. The method of claim 51 wherein the protected, unsaturated hexopyranose is produced by:

59. The method of claim 58 wherein the protected, unsaturated hexopyranose is produced by:

60. The method of claim 42 wherein glycosylation of the nucleobase proceeds as :

wherein B is a free or protected heterocyclic base.

61. The method of claim 60 wherein glycosylation of the nucleobase proceeds as :

62. The method of claim 39 wherein deprotection of the protected oxepine nucleoside proceeds as:

63. The method of claim 62 wherein deprotection of the protected oxepine nucleoside proceeds as :

64. The method of claim 35 wherein reduction of the oxepine nucleoside proceeds as:

65. The method of claim 64 wherein reduction of the oxepine nucleoside proceeds as:

66. The method of claim 35 further comprising the steps of protecting the oxepane nucleoside and phosphitylating the protected oxepane nucleoside to yield a protected oxepane nucleoside phosphoramidite derivative for use in oligonucleotide synthesis .

67. The method of claim 66, wherein the oxepane nucleoside is protected at a hydroxyl group by tritylation.

68. The method of claim 67 wherein the tritylation and phosphitylation proceed as:

69 . The method of claim 67 wherein the steps of tritylation and phosphitylation proceed as :

70. A phosphoramidite derivative comprising the nucleoside of any one of claims 1 and 11.

71. Use of the oxepane nucleoside of any one of claims 1 and 11 for synthesis of oligonucleotides, wherein the oxepane nucleoside is subjected to tritylation and phosphitylation prior to synthesis.

72. A compound selected from the group consisting of compounds of formulas (VII) , (VIII) , (IX) and (X) :

wherein B is selected from the group consisting of thymine, uracil, cytosine, adenine, guanine, inosine, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, isocytosine, N⁴-methylcytosine, 5-iodouracil, 5-fluorouracil, 4-thiouracil, 2-thiouracil, (E) -5- (2 -bromovinyl) uracil, 2-thiothymine, 4- thiothymine, 7-deazaadenine, 9-deazaadenine, N⁶-methyladenine, 2-chloroadenine, 2-fluoroadenine, 2-chloroadenine, isoguanine, 3-deazaadenine, 7-deazaguanine, 9-deazaguanine, 6-thioguanine, nicotinamide, 2-aminopurine, 2, 6-diaminopurine, N6-cyclopropyl- 2 , 6-diaminopurine, hypoxanthine , 6-thiohypoxanthine, and 1,2,4- triazole-3-carboxamide, and

mirror image enantiomers thereof.